Mitglied der Helmholtz-Gemeinschaft

BioSoft

Selected results 2007/2008

Contents

Editorial Introduction Selected Results

Institute of Solid State Research (IFF)
Theoretical Soft Matter and Biophysics Neutron Scattering Soft Matter

page

page

page page page page page page page

7 9 27 43 59 73 89

Institute of Structural Biology and Biophysics (ISB)

Cellular Biophysics Molecular Biophysics Structural Biochemistry

Institute for Bio- and Nanosystems (IBN)

Bioelectronics Biomechanics page 105 page 115 page 129

Education & Dissemination Publications

page 139

Impressum BioSoft Selected Results 2007/2008 Herausgeber: Forschungszentrum Jlich GmbH Stabsstelle Fachstrategie Institut fr Festkrperforschung 52425 Jlich Deutschland +49-2461-61-3026 +49-2461-61-3570 www.fz-juelich.de Redaktion: Dr. Wolfgang Speier Kurt Wingerath Druck: Grafische Medien Forschungszentrum Jlich GmbH

Juni 2009 Forschungszentrum Jlich GmbH

Editorial

Physics meets Biology

Physicists and Biologists worldwide have recognized some time ago that they can benefit a lot from sharing their knowledge and working together to understand the behavior of complex systems. At the Forschungszentrum Jlich, physicists and biologists have started to intensify their collaboration in 2004 by organizing a two-week IFF spring school with the title Physics meets Biology. This school was attended by an international audience of about 200 students. We are very happy that Erwin Neher, the Nobel laureate and inventor of the patch-clamp technique (together with Bert Sakmann), was among the lecturers of this school. The success of this spring school made clear to us that a more long-term effort is needed to provide an interdisciplinary graduate education, in order to equip students and young researchers with the necessary techniques and knowledge to address the many exciting scientific challenges at the interface between biology, chemistry and physics. This has lead the foundation of the International Helmholtz Research School on Biophysics and Soft Matter (IHRS BioSoft), together with colleagues from the Universities of Dsseldorf and Kln, supported by a grant from the Helmholtz Association. The school accepted its first students in the fall 2006. The research center CAESAR in Bonn has joined the graduate school recently. In parallel, we have continued to educate a wider range of students by organizing spring schools, in particular in 2008 by a school entitled ``Soft Matter From Synthetic to Biological Materials". Research in Soft-Matter and Biophysics spans over three institutes on Jlich campus: the Institute of Solid State Research (IFF), the Institute for Bio- and Nanosystems (IBN) and the Institute of Structural Biology and Biophysics (ISB). Scientist from these institutes share many common interests, jointly use modern experimental techniques and instrumentation, and work on several joint research projects. The current brochure presents a selection of reports, which illustrate the research in SoftMatter and Biophysics in the Forschungszentrum Jlich during the last two years.

Gerhard Gompper

Introduction
BioSoft Biophysics & Soft Matter
Macromolecules consisting of hundreds to thousands of atoms have many emergent properties, which are not present in small molecules. Prominent examples are linear polymers, which are constructed from a single or a few kinds of building blocks. Even simple synthetic polymers that are typically made from identical monomers allow the design of complex material properties that depend, for example, on their lengths, building blocks, cross linking, and potential combination with other polymers. Highly advanced methods often requiring large-scale facilities are used to investigate their fascinating properties. Also, the molecules of life, nucleic acids and proteins, are linear chain molecules, consisting of four and twenty different building blocks, respectively, in a strictly defined linear sequence. Thus, it is obvious that biological macromolecules have much more complex properties, the investigation of which requires the most advanced and due to production limits - most sensitive methods available. Many interesting properties of macromolecules are based on their complex interactions among each other. This is especially true for the extremely specific interactions between proteins. Biophysics and soft matter physics are concerned with the qualitative and quantitative description of structure and dynamics of complex macromolecules and their assemblies at various levels up to living cells.

Biophysics

seeks to understand the processes of life by applying advanced physical methods to complex biological systems. This understanding requires exquisitely detailed and quantitative knowledge of the underlying processes at the molecular, cellular, and systems level. The development and fate of every single cell is regulated on the molecular level by a variety of cross-linked mechanisms. Therefore, structural biology aims at obtaining precise geometric and dynamic information about biologically and medically relevant molecules as a basis for an understanding of their molecular functions. On the cellular level, the efficiency of interlinked signaltransduction chains and of information processing is enormously high. By interaction with their environment, cells constantly receive, process, and emit information such as chemical, mechanical and electrical stimuli. Here, a detailed understanding is very important, because malfunctions of cellular information processing caused by endogenous or exogenous factors, e.g., spontaneous mutations, pathogens or environmental influences, can lead to serious diseases or to accelerated ageing processes.

Soft Matter

encompasses all kinds of macromolecular assemblies of polymeric, colloidal, or amphiphilic character. The aim of Soft Matter research is to understand the cooperative behaviour of macromolecular systems with many interacting degrees of freedom, and to provide the scientific basis for a rational design of functional nano-scale materials. Thermodynamics drives the selfassembly of macromolecular building blocks into mesoscopic structures with a large variety of rheological, electronic, photonic, magnetic, thermal, or mechanical properties. Due to the weak interactions between building blocks, thermal fluctuations compete with conservative forces arising, e.g., from van der Waals or Coulomb interactions, and with topological constraints. Large mesoscopic length scales imply long time scales, and slow dynamical behavior. Therefore, nonequilibrium behavior is ubiquitous in soft matter systems driven by external fields or hydrodynamic flows. This implies that such fields can be employed to modulate, assist, or direct structure formation, and thereby taylor material properties.

Worldwide, we experience a rapid convergence of physical and biological research areas and possibly also research structures. Soft matter physics and biophysics represent a very dynamic and rapidly growing area of multidisciplinary research at the interface between physics, chemistry and biology. This requires a highly multidisciplinary infrastructure combining medium and large-scale experimental facilities with scientific computing and a high-level laboratory science. There are several compelling reasons for this approach: A profound understanding of fundamental mechanisms underlying biological function and self-organisation can be fostered by studies of simpler synthetic soft matter systems. Biological components or biological construction principles can be used or mimicked in the design of new materials. Advanced physical tools and quantitative methods provide new routes in the investigation of biological molecules and systems. A profound, quantitative understanding of selected model systems ranging from molecules to cells lays the foundations for modern systems biology. Knowledge and techniques created by cutting-edge research in this transdisciplinary field is essential for the sustained rapid progress in nanotechnology, especially soft nanoscience, nanobiotechnology, materials sciences, and life and health sciences.

Selected Results

Institute of Solid State Research (IFF)

Theoretical Soft Matter and Biophysics Neutron Scattering Soft Matter

Institute for Structural Biology and Biophysics (ISB)

Cellular Biophysics Molecular Biophysics Structural Biochemistry

Institute of Bio- and Nanosystems (IBN)

Bioelectronics Biomechanics

2007 2008
7

IFF-2: Theoretical Soft-Matter and Biophysics

Director: Prof. Gerhard Gompper

The main research topic of the Institute Theoretical Soft Matter and Biophysics is the theory of macromolecular systems. Soft matter physics and biophysics are interdisciplinary research areas encompassing statistical physics, materials science, chemistry, and biology. Our systems of interest include polymer solutions and melts, colloidal suspensions, membranes, vesicles and cells, but also composite systems ranging from colloids in polymer solutions to mixtures of surfactants and amphiphilic block copolymers. A major focus is the hydrodynamic behaviour of complex fluids and biological systems, both in equilibrium and under flow conditions.

At IFF-2, a large variety of methods are applied. In fact, a combination of analytical and numerical methods is often required to successfully characterize the properties of these complex systems. In particular, simulation methods (Monte Carlo, molecular dynamics), mesoscale hydrodynamic simulation techniques, field theory, perturbation theory, and exact solutions are employed. Since the building blocks of soft matter systems often contain a large number of molecules, simplified mesoscale modelling is typically required, which is then linked to the molecular architecture. A characteristic feature of soft-matter research is the fruitful interaction between theory and experiment. IFF-2 closely cooperates with the Institute for Neutron Scattering (Prof. Richter) and the Institute for Soft Condensed Matter (Prof. Dhont) to successfully tackle many of the essential aspects of the systems investigated.

Relevance of Angular Momentum Conservation in Fluid Simulations

I. O. Gtze , H. Noguchi , G. Gompper
IFF-2: Theoretical Soft-Matter and Biophysics

The angular momentum is conserved in uids with a few exceptions such as ferrouids in external elds. However, it can be violated locally in uid simulations to reduce computational costs. The effects of this violation are investigated using multi-particle collision dynamics, a well-established, highly efcient hydrodynamics simulation technique, where an angularmomentum conserving variant is available. We show that there are situations of practical relevance, such as multi-phase ows in Couette geometry, where angular momentum conservation is essential to avoid non-physical results. In simulations of the hydrodynamic behavior of complex uids, one is faced with the challenge of bridging the gap between the mesoscopic length and time scales of the solute and the atomic scales of the solvent. As these typically differ by orders of magnitude, a full treatment on a microscopic level is prohibited by the enormous number of involved particles and the large necessary time range. Moreover, often only the dynamics of the colloidal particles are of particular interest, while the microscopic details of the solvent that mediates the hydrodynamic interactions are rather unimportant. Thus, a coarse-grained mesoscopic uid model is required that is sufciently simple to be tractable but still captures the correct hydrodynamic behavior. By representing a large number of physical solvent molecules by one model uid particle at a time, the number of degrees of freedoms can be reduced considerably. Various mesoscopic approaches have been proposed in the last decades, which can be categorized in lattice methods or particle-based methods. We focus on particlebased simulations, where particle positions and velocities are continuous variables that are updated at discrete times. Here, coupling to solute particles as well as moving boundaries can be treated more easily than in lattice methods and thermal uctuations are naturally contained. Moreover, lattice methods generally suffer from the lack of Galilean invariance. The method used here, multi-particle collision dynamics (MPC) [1], needs less computational time compared to other particle based methods such as DPD, thus allowing simulations of larger systems. Here, the uid is represented by point particles that undergo subsequent streaming and collision steps. In the streaming step, the particles move ballistically.

Subsequently, they are sorted into collision cells and the collision step then mimics the simultaneous interaction of all particles within each cell by assigning them new velocities. MPC has been applied to various systems such as colloids, polymers, membranes, ternary amphiphilic uids, and chemical reaction systems. Hybrid simulations combining an MPC uid with molecular dynamics of solute particles are easily possible. The algorithm is constructed in such way that mass, energy and translational momentum are locally conserved, which is essential for correct hydrodynamic behavior. However, the angular momentum is not conserved in the most widespread version of MPC, which is often called stochasticrotation dynamics. In order to clarify the effects of angular-momentum conservation [2], we mainly use the Andersen-thermostat version of MPC, where angular momentum conserving (MPC-AT+a) and nonconserving (MPC-ATa) algorithms are available [3]. In conventional viscous uids that do conserve angular momentum, the viscous stress tensor has to be symmetric, i. e. = . This symmetry is required by the fact that there is no stress expected in a uniformly rotating uid (rigid body rotation), or alternatively, by the conservation of angular momentum. On the other hand, for a uid without conservation of angular momentum, the above argument is no longer valid and we have to consider in general an asymmetric tensor. Then, the viscous stress is given by = + ( v) (1) v v v v + + , x x x x

where , {x, y, z}. Here, is the second viscosity coefcient, and and are the symmetric and asymmetric components of the viscosity, respectively. The last term in Eq. (1) is linear in the vorticity v, and does not conserve angular momentum. Thus, the last term vanishes (i. e. = 0) in angular momentum-conserving systems. The equation of velocity evolution is given by Dv = P +(+ ) ( v)+(+ ) Dt
2

v, (2)

where D/Dt is Lagranges derivative and P is the pressure. When a uid is incompressible, this is the normal Navier-Stokes equation with viscosity = + . Since the equations of continuity and veloc ity evolution are of the same form, the negligence

10

of angular-momentum conservation does not modify the velocity eld of uids when the boundary conditions are given by velocities. However, it generates an additional torque, so that the velocity eld can be changed when the boundary condition is given by forces. In cylindrical coordinates (r, , z), the azimuthal stress is given by r = ( + ) r(v /r) v + 2 . r r (3)

The rst term is the stress of the angular-momentumconserving uid, which depends on the derivative of the angular velocity = v /r. The second term is the additional stress from the negligence of angularmomentum conservation and is proportional to . We consider a uid conned between two coaxial cylinders (Couette ow) rotating with the same angular frequency . Here, no torque is expected to be acting on the cylinders in angular-momentum conserving uids, as this corresponds to the rotation of a rigid body. However, in simulations without angularmomentum conservation, we do observe torques on the conning cylinders, induced by the stress term of the asymmetric viscosity in Eq. (3). The torque is the tangential stress 20 multiplied by the circum ference length 2R and the radius R. The nite size of the collision cells leads to a correction term, so that the torques on the conning cylinders are found to be 3a Tin,out (R) = 4 R2 1 . (4) 4R In order to verify that the velocity eld is not affected by the lack of angular-momentum conservation if the boundary condition is given by the velocity, we study uids conned between eccentric cylinders with xed axes, where the inner one is rotating at constant angular velocity. As expected, the corresponding stream lines, which are shown in Fig. 1, are practically identical for both methods. The occurrence of a back-ow in the large-gap region as well as the resulting total forces on the inner cylinder are in good agreement with theoretical predictions.

FIG. 2: Azimuthal velocity v of binary uids in a rotating cylinder. The uids with different viscosities are located at r < 5a and 5a < r < 10a, respectively. Symbols represent the simulation results of MPC-ATa for two different viscosity ratios (+, ), and MPC-AT+a (). Solid lines represent the analytical results for MPC-ATa.

in MPC-ATa uids, we consider binary uids with a xed geometry of the boundary surface, which is impenetrable to the uid particles. The inner cylinder of radius R1 of circular Couette ow is replaced by a more viscous uid, and the outer cylinder with radius R2 rotates with constant velocity 0 . This is a simplied description of oil and water phase-separated due to surface tension, or two liquids separated by a membrane. It is assumed that the cylinder rotates very slowly, and that the ow stress does not change the shape of the interface. We choose the uid inside (r < R1 = 5a) to have a higher viscosity than the uid outside. The MPC collision performed in cells crossing the boundary propagates the momentum from one uid to the other. In MPC-AT+a, both uids rotate with 0 independent of their viscosities. However, in MPC-ATa, the inner uid rotates more slowly (see Fig. 2). This is caused by the asymmetric stress term 2. If both uids rotate at the same angular velocity, the inner and outer stresses do not coincide. The theoretical velocity prole is obtained from the stress balance at r = R1 and is in very good agreement with the numerical results (Fig. 2). Our main conclusion is that simulations which do not conserve angular momentum can lead to quantitatively and even qualitatively incorrect results, when the boundary conditions on walls are given by forces, uids with different viscosities are in contact, or nitesized objects rotate in uids.

[1] A. Malevanets and R. Kapral, J. Chem. Phys. 110, 8605 (1999). FIG. 1: Stream lines for uid conned between eccentric cylinders (inner one rotating with constant angular velocity). [2] I. O. Gtze, H. Noguchi, and G. Gompper, Phys. Rev. E 76, 046705 (2007). [3] H. Noguchi, N. Kikuchi, and G. Gompper, Europhys. Lett. 78, 10005 (2007).

A boundary of a uid does not only exist on solid objects but also between two uids or on membranes. In order to investigate the uid-uid boundary

11

Diffusion and Segmental Dynamics of Double-Stranded DNA

R. G. Winkler 1 , E. P. Petrov 2 , T. Ohrt 2 , P. Schwille 2
1 2

IFF-2: Theoretical Soft-Matter and Biophysics Institute of Biophysics/BIOTEC, Dresden University of Technology, Dresden

The diffusion and segmental dynamics of doublestranded -phage DNA molecules are quantitatively studied over the transition range from stiff to semiexible chains. Spectroscopy of uorescence uctuations of single-end uorescently labeled monodisperse DNA fragments unambiguously shows that double-stranded DNA in the length range of 102 2 104 base pairs behaves as a semiexible polymer with segmental dynamics controlled by hydrodynamic interactions. The dynamic behavior of individual macromolecules in solution is governed by chain connectivity and hydrodynamic interactions [1]. Understanding of polymer dynamics and quantitative verication of polymer theories require detailed information on segmental motion of individual polymer molecules. However, the classical experimental techniques, such as dynamic light scattering (DLS) or transient electric birefringence (TEB), predominantly deliver information on large-scale shape uctuations of macromolecules. Fluorescence correlation spectroscopy (FCS) [2] is a single-molecule technique that can provide more detailed information on the macromolecular dynamics than the classical ensemble-based methods. By uorescent labelling of individual segments [3] or continuous labelling of the whole molecule [4], the diffusional motion of segments as well as that of the overall molecule can be studied at nanomolar concentration under (quasi)equilibrium conditions in solution and cellular systems. Precise experiments in polymer physics are impossible without well-dened monodisperse polymer samples covering a wide range of molecular weights. The recent progress in molecular biotechnology resulted in a variety of techniques to produce monodisperse DNA fragments, which stimulated the use of DNA as a model compound in studies of polymer dynamics in solution. We employed the technique of polymerase chain reaction (PCR) to produce monodisperse samples of DNA fragments with predened sequence, structure, and length, uorescently labeled at the same single end [3]. Double-stranded (ds) DNA is a biopolymer characterized by a large persistence length lp 50 nm [3]. As a result, dsDNA fragments exhibit rodlike, semiexible, or even exible polymer behavior, depending on their length. Thus, simple generic models of polymer dynamics [1]

are not expected to provide a quantitative description of dsDNA behavior, and more advanced models, accounting for the persistence of the polymer chain, e.g., the semiexible polymer model of Ref. [4], are required. Recent FCS studies raised the question whether dsDNA dynamics in dilute solution is controlled by hydrodynamic interactions [4] or not [5]. Moreover, no experimental studies were reported previously, where diffusion and intramolecular dynamics of dsDNA had simultaneously been investigated over the transition range from stiff to semiexible chains. Our FCS measurements of lengths L from 102 to 2104 base pairs (bp) (L/lp 0.7 140) by following the Brownian motion of the labeled ends of single DNA molecules lled this gap and showed that the experimental data can quantitatively be described by the theory for semiexible polymer dynamics [3], which clearly demonstrates that in this length range dsDNA behaves as a semiexible polymer with strong hydrodynamic interactions. In FCS [2], uctuations of the confocally detected uorescence signal F (t) = F + F (t) are studied via the correlation function G ( ) = F (t) F (t + ) / F 2 . In the absence of additional photophysical processes and chemical reactions, the uorescence signal uctuates as a result of the Brownian motion of uorescently labeled particles through the detection volume whose shape can be approximated by a 3D Gaussian ` 2 2 exp 2r2 /r0 2z 2 /z0 , where r0 and z0 are the lateral and axial extensions of the detection volume, respectively. With the mean square displacement (MSD) r2 (t) of a particle, the FCS correlation function assumes the form 1 1 G (t) = , (1) r N r2 (t) r2 (t) 1 + 2 z2 1 + 2 r2 3 3
0 0

where N is the effective number of molecules in the detection volume. Utilizing the Gaussian semiexible polymer model, analytical calculations yield the MSD 2 2kB T X 2 r (t) = 6Dt+ n n (L/2) 1 et/n n=1 (2) for the chain end [4], where T is the temperature, kB the Boltzmann factor, and the viscosity. n (L/2)

12

FIG. 1: Normalized FCS correlation functions (top) and determined mean square displacements (bottom) for -DNA fragments of lengths 0.1, 0.2, 0.5, 1, 2, 5, 10, and 20 kbp (from left to right). The intramolecular contribution to the MSD is shown by the green lines (bottom). The red lines indicate the power laws r2 t and t2/3 , respectively.

FIG. 2: Diffusion coefcients (top) and longest relaxation times (bottom) of dsDNA. The blue symbols indicate our FCS data. The other data (black symbols) are taken from various sources [3]. The red lines are predictions of the semiexible polymer model [3, 4] with the parameters lp = 50 nm, D = 0.9, and = 0.6.

is the value of the n-th eigenfunction of a chain of length L at its end, n is the n-th relaxation time in the presence of hydrodynamic interactions and is related with the free-draining relaxation time n via n = n /(1 + 3Hnn ). D is the translational dif fusion coefcient of the macromolecule, and Hnn is the matrix element of the Rotne-Prager tensor within the preaveraging approximation [4]. Here, and D are t parameters, which are introduced in our quantitative analysis of the experimental data [3]. For long and exible molecules, L/lp 1, the theoretical 2 expression yields the short-time behavior r (t) t , with the exponents = 1/2 (free draining) and 2/3 (nondraining), in agrement with predictions of the Rouse and Zimm model, respectively [1]. For semiexible polymers, the short-time behavior is given by = 3/4 [4]. However, these power laws are only valid for times much shorter than the longest relaxation time of the polymer, whereas at comparable and longer times the intramolecular contribution to the MSD saturates due to the nite size of the polymer coil, as shown in Fig. 1. Thus, direct application of the power laws to the analysis of the MSD time dependencies, as has been proposed recently in Ref. [5], can be misleading. Indeed, even for the longest sample the apparent power law in r2 (t) observed in the range of 100 105 s is to a large extent due to the crossover from intramolecular motion to overall polymer diffusion; only for times shorter than 103 s a power law-type behavior can observe in the intramolecular contribution. We would like to point out that such an analysis of the shorttime dynamics, still possible in our study for the two longest DNA fragments, clearly rules out the Rousetype behavior reported for dsDNA in Ref. [5] and leads to a denitive conclusion on the importance of intramolecular hydrodynamic interactions in dsDNA polymer dynamics. Figure 2 displays diffusion coefcients and longest relaxation times the upper part is not readily acces-

sible by DLS or TEB which we obtained by tting of the normalized FCS curves [3]. The experimental correlation functions are very well reproduced for the parameters lp = 51 1 nm, = 0.6, and D = 0.9. Within the range of lengths studied, the diffusion coefcients and relaxation times exhibit approximately the power law behavior D L2/3 (note that there is no excluded volume interaction for short DNAs) and r L1.7 , and thus do not follow the predictions of the Rouse (D 1/L, r L2 ) nor the Zimm (D 1/L1/2 , r L3/2 ) model. At the same time, our data are in very good agreement with results obtained by other experimental techniques not related to FCS [3] and closely follow the predictions of the semiexible polymer theory [4]. Therefore, our results clearly demonstrate that in the range of the lengths studied, dsDNA behaves as a semiexible polymer with strong hydrodynamic in teractions . The Zimm regime with r2 t2/3 , 1/2 3/2 D L , and r L (or corresponding expressions modied to account for excluded-volume interactions) [1] can be achieved only for long dsDNA molecules with lengths exceeding 105 bp, or 103 lp (Fig. 2).

[1] M. Doi, S. F. Edwards, The Theory of Polymer Dynamics (Clarendon Pres, Oxford, 1986) [2] E. P. Petrov, P. Schwille, State of the Art and Novel Trends in Fuorescence Correlation Spectroscopy (Springer, Berlin, 2007) [3] E. P. Petrov, T. Ohrt, R. G. Winkler, P. Schwille, Phys. Rev. Lett. 97, 258101 (2006) [4] R. G. Winkler, S. Keller, J. O. Rdler, Phys. Rev. E 73, 041919 (2006) [5] R. Shusterman, S. Alon, T. Gavrinyov, O. Krichevsky, Phys. Rev. Lett. 92, 048303 (2004)

13

Biological Nanomachines
A. Baumgrtner, J.-F. Gwan, S. Grudinin, M. Haan
IFF-2: Theoretical Soft-Matter and Biophysics

Biomolecular machines are large protein complexes whose activities are essential for providing life to a biological cell. Among the various known biomolecular devices, ion channels, which reside in cell membranes, are one of the simplest bionanomachines. We have investigated using computer simulations the molecular mechanism of selectivity and transport of ions through a specic potassium ion channel.

of the atomic structures and forces involved. Although a complete description is not yet available even for the best-characterized system, considerable progress has been made recently, not only from an experimental point of view, but also with respect to computational and theoretical achievements. One of the most fascinating biomolecular machines is the class of vacuolar H+ -ATPases (or V-ATPases), which are a family of ATP-dependent proton pumps responsible for acidication of intracellular compartments and proton transport across the cell plasma membrane. V-ATPases are multisubunit complexes (Fig.1) composed of a peripheral domain V1 (yellow and orange) responsible for ATP hydrolysis and an integral domain V0 (blue and grey) responsible for proton translocation. The V-ATPases are thought to operate by a rotary mechanism in which ATP hydrolysis in V1 drives rotation of a ring of proteolipid subunits in V0 . The V1 and V0 domains are connected by both a central stalk which is thought to play a crucial role in the assumed rotary mechanism of ATP-driven proton transport. The molecular details at atomic resolution are only partly resolved. The theoretical understanding are currently beginning to emerge. This molecular machine is under investigation using elaborate modelling and computer simulations.

FIG. 1: Cartoon representation of the V-ATPase, a biological nanomachine which acts as a ATP-driven proton pump.

Nanotechnology is perfectly realized in biological systems. Cells are essentially biological assemblers that build thousands of custom-designed molecules and construct new assemblers. This view was pioneered by Richard Feynmans [1] evocative idea of a self-replicating assembler building nanoscale devices atom by atom. Living cells are made up of these complexes, which carry out many of the functions essential for their existence, differentiation, and reproduction. In many cases the malfunction of these proteins can be a source of disease. Many of these complexes can be described as molecular machines or molecular motors or molecular devices, depending on their sizes, complexity and tasks [2]. The essential question in understanding biomolecular machines is concerned with the explanation of the macroscopic phenomenology in terms

FIG. 2: Structure of the KcsA potassium channel embedded in a water solvated lipid bilayer membrane. Only two monomers of the tetrameric protein are shown.

14

A much simpler and much more well characterized bionanomachine is the KcsA potassium channel [3], which acts as a valve in the plasma membrane of prokaryotic cells. The cutaway view of the atomic structure of the protein complex (in ribbon representation) is shown in Fig.2. The gure shows only two monomers of the tetrameric protein complex embedded in a water solvated lipid bilayer membrane. Each monomer consists of three helices, one extracellular loop, and as the central part of the channel the selectivity lter containing three potassium ions (depicted by balls) and one water molecule between. The structural details of the lter are decisive for ion selectivity and transport. The selectivity lter facilitates the diffusion of potassium ions at rates approaching 108 ions per second under physiological electrochemical gradients. The ability of potassium channels to conduct several K+ ions simultaneously in a single le through the narrow pore at levels near the limit of diffusion is usually described in terms of concerted mechanisms. It has been suggested that to reach a high conduction rate in a long-pore channel, ions must move through the channel pore in a multi-ion fashion : the permeating ions line up in the narrow channel pore and move in a single le through the channel. This is known as the multi-ion permeation process, and is believed to be a common feature of the ion transportation process in all potassium channels. The multi-ion theory has been acepted over decades, but the molecular mechanism of it remained elusive.

three distinct features of the molecular mechanism. (1) The movements of neighboring ions and water molecules are strongly correlated. They mostly moved in pairs or triples (permons). One important fact for the understanding of the efciency of the correlated movements is that the periodicity of the potential in the selectivity lter, 3 , practically matches with the distance of the lowest Coulomb energy UKW (z) between one ion and one water molecule. The classical potential energy UKW (z) between a single ion and a single water molecule at distance z, consisting of Coulomb and van der Waals interactions, exhibits energy minima at characteristic distances of z 3 (bound states). The depth of the potential depends on the orientation of the water molecule with respect to the ion. (2) Permons are polarized. The absolute minimum of UKW (z) 30 kB T is at distance z = -2.65 and when the oxygen of the water dipol is oriented towards the ion (polarized bound state). When the outermost ion exits the channel to the periplasmic side, the next neigboring ion hops to this vacancy, the ions neighboring water molecule prefers to maintain its bound state and follows almost simultaneously the preceding ion towards the vacancy. This is the movement of a permon. (3) The movements of permons are rectied. Another interesting observation from our MD simulations is that very rarely a water molecule has been observed to hop back to the vacancy and join the other ion to form a bound state. The absence of such an event is explained by the energy barrier imposed by the periodic pore potential on the water molecule. This implies that a water molecule acts as a pawl in a ratchet mechanism. One implication is that the transport of ion-water pairs through an ion channel is much more efcient than the transport of ions only. In summary, the high permeation rate at which ion and water molecules pass through the KcsA ion channel is based on the cooperative hopping of pairs of ions and water molecules mediated by the exible charged carbonyl groups lining the backbone of the channel. These observations provide the basis of an atomistic concept of the molecular mechanism of the multi-ion transport mechanism.

FIG. 3: Molecular view of the selectivity lter of the KcsA potassium channel including ions and water molecules. [1] R. P. Feynman, in Miniaturisation, page 282-296, ed. by H. D. Gilber, Reinhold, New York, 1961.

Employing MD simulations on the basis of the Xray structure of the KcsA channel, our studies [4, 5] have provided useful insights into the structureconductivity relationship. By pulling out the outermost ion from the exit of the pore (Fig.3), we observed subsequent collective cooperative movements of ions, water and carbonyl groups lining the backbone of the pore. A detailed analysis of these movements lead to the development of a simple twodimensional model of ion-water transport. Based on our molecular dynamics simulations [4, 5] we found

[2] A. Baumgaertner, in Handbook of Theoretical and Computational Nanotechnology, ed. by M. Rieth and W. Schommers, Am. Sci. Publ., 2006. [3] D.A.Doyle, J. Morais-Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S .L Cohen, B. T. Chait and R. MacKinnon, Science 280, 69 (1998). [4] J. F. Gwan and A. Baumgaertner, J. Chem. Phys. 127, 045103 (2007). [5] J. F. Gwan and A. Baumgaertner, J. Compt. Theor. Nanoscience 4, 50 (2007).

15

Diffusion in a uid membrane with a exible cortical cytoskeleton

T. Auth 1 , N. S. Gov 2
1 2

IFF-2: Theoretical Soft-Matter and Biophysics The Weizmann Institute of Science, Department of Chemical Physics, P.O. Box 26, Rehovot 76100, Israel

The cytoskeleton hinders protein diffusion in the lipid bilayer of the cells plasma membrane. We calculate the inuence of a exible network of long-chain protein laments, which is sparsely anchored to the bilayer, on protein diffusion. We dene a potential landscape for the diffusion based on the steric repulsion between the cytosolic part of the protein and the cytoskeletal laments, using the pressure eld that the cytoskeleton exerts on the bilayer. We predict the changes of the diffusion coefcient upon removal of anchor proteins and for a stretched cytoskeleton. Diffusion within the uid membrane plays an important role for cellular processes, because the cell communicates with its surrounding via its lipid bilayer [1]. For example, diffusion of activated receptor molecules leads to signal amplication and diffusion of adhesion molecules to the contact area is important for cell adhesion. The cytoskeleton, which is in close proximity to the membrane, has been identied to act as a strong regulator of the diffusion within the cellular membrane. For the red blood cell (RBC), several experiments show that the spectrin cytoskeleton slows down translational diffusion. Using single-molecule tracking techniques, small compartments have been found for the diffusion in the cell membrane [2] that may be explained by the cytoskeletal network below the bilayer. Our work focusses on the diffusion in the membrane of cells that have a cortical, two-dimensional cytoskeleton, which is composed of exible long-chain proteins [3]. A prominent example of such cells are RBCs, where the long-chain proteins are spectrin tetramers. However, a similar cortical cytoskeleton is found on the plasma membrane of other mammalian cells, and spectrin has been identied in neurons and on membranes of intracellular organelles. A pressure eld on the bilayer is generated by the conformational uctuations of the cytoskeletal laments to which they are anchored at their ends via anchor complexes [4] . We calculate the pressure eld using a linear, exible polymer with bulk radius of gyration Rg that is attached to a hard wall at 1 = (x1 , y1 ) and 2 = (x2 , y2 ). The pressure diverges close to the anchor points and decreases

FIG. 1: Entropic pressure that a exible, cytoskeletal protein that is anchored to a lipid bilayer with its ends exerts on the cell membrane.

exponentially for large distances from the anchors, || |i | (i = 1, 2), see Fig. 1. The calculation of the pressure is based on the diffusion equation to calculate the polymer conformations. This implies an innite contour length of the polymer, which is certainly a bad approximation if the distance d between the anchor points is comparable with the contour length. This situation is addressed in Ref. [5], where a Gaussian polymer model is combined with the condition of a nite contour length. Force-extension relations and end-to-end distribution functions have been calculated and the analytic theory describes experimental and simulation data very well. We use a simple argument to illustrate how a nite contour length affects the uctuation pressure: we 2 subtract from the contour length L = 6Rg,0 / (where = L/N is the Kuhn length of the polymer, N the number of repeat units in the chain, and Rg,0 the radius of gyration of the free chain) the anchor distance d, because this length of the chain is needed to connect the anchor points and is therefore not available for conformation uctuations. To calculate the pressure eld of the stretched chain, we simply replace Rg in the expression for the pressure by the new, effective Rg , which we obtain using 2 the effective Kuhn length, = (L d)/N : Rg = 2 N/6 = ( /6)(L d)2 /L. The pressure along the connection line of the anchor points is large either for very small anchor distances because of the high pressure at the anchor points or for very large an-

16

FIG. 2: Pressure eld for an unstretched RBC cytoskeleton.

FIG. 3: Anisotropic diusion for a cytoskeleton that is stretched by a factor 1.5 and accordingly compressed in the perpendicular direction (for two dierent orientations of the hexagonal network).

chor distances that are comparable with the countour length when the polymer conformation is stretched and therefore everywhere close to the lipid bilayer. Fig. 2 shows a superposition of the single-lament pressure elds for an idealized arrangement of spectrin bonds on a hexagonal lattice, as in the RBC cytoskeleton. We use a random walk and Metropolis Monte Carlo to simulate the diffusion process in the lipid bilayer. The step length is a = 1 nm, which is the typical size of the lipid molecules. The inuence of the cytoskeleton is taken into account by the potential landscape that is determined for the pressure eld of the cytoskeleton multiplied with the effective interaction volume for the protein under consideration. The interaction volume is determined by the size of the cytosolic part of the protein that sterically interacts with the cytoskeleton. It can be obtained either from electron microscopy data for the spectrin and the diffusing protein or phenomenologically as a t parameter using measurements of the effective diffusion coefcient. Once the effective interaction volume is determined, we can predict several aspects of the protein diffusion [3]. For short times, the protein shows normal, fast diffusion within a single corral that is formed by the cytoskeletal bonds. Also for long times normal diffusion is observed, but with a smaller, effective diffusion constant. Long-time diffusion is hindered because the protein needs to hop across the potential barriers. The crossover between both regimes can also be observed in single-particle tracking experiments. In healthy red blood cells, the cytoskeletal laments are usually attached at their ends but also with a different protein complex in their middle. In disease, the middle anchor complex can be missing. We predict that the diffusion coefcient in the disease case is by a factor 25 larger than for healthy RBCs. Furthermore, we can predict the inuence of compression or stretching of the cytoskeleton on protein diffusion. For isotropic stretching, we nd a decrease of the effective diffusion constant because of the increased height of the cytoskeletal barriers. For the normal RBC, the simulation results are well de-

scribed by a rescaled analytic expression: the height of the potential barrier is given by the value in the middle of the bond where the cytoskeletal pressure is lowest. For anisotropic stretching, which constantly occurs in vivo when the RBC is deformed in blood ow, we predict increased diffusion along the direction of the stretch and decreased diffusion perpendicular to the stretching. In Fig. 3, we plot the relative diffusion for an anisotropically stretched network parallel and perpendicular to the direction of the stretch for different values of the effective interaction volume. For the effective interaction volume v = 6000 nm3 that applies for the band-3 protein in RBCs, we nd that the diffusion is almost completely asymmetric if the RBC cytoskeleton is stretched by a factor 1.5 and accordingly compressed in the perpendicular direction (to preserve the total area, which is xed by the lipid bilayer). Our results quantify the interaction between the cytosolic part of a transmembrane protein and a cytoskeleton that consists of a two-dimensional network of exible polymers, as for example found in RBCs. We predict the changes in the diffusion due to lack of anchor proteins, such as a missing middle anchor complex in forms of hereditary spherocytosis. We also predict changes of the diffusion caused by a stretched cytoskeleton.

[1] M. J. Saxton, Curr. Top. Membr. 48, 229 (1999). [2] M. Tomishige, Y. Sako, and A. Kusumi, J. Cell Biol. 142, 989 (1998). [3] T. Auth and N. S. Gov, Biophys. J. 96, 818 (2009). [4] T. Auth, S. A. Safran, and N. S. Gov, New J. Phys. 9, 430 (2007). [5] R. G. Winkler and P. Reinecker, Macromolecules 25, 6891 (1992).

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Attractive colloidal rods in shear ow

M. Ripoll, R. G. Winkler, G. Gompper
IFF-2: Theoretical Soft-Matter and Biophysics

Suspensions of rod-like colloids show in equilibrium an isotropic-nematic coexistence region, which depends on the strength of an attractive interaction between the rods. By means of hydrodynamic simulations, we study the behavior of this system in shear ow for various interaction strengths. The shear ow induces alignment in the initially isotropic phase which generates additional free volume around each rod and causes the densication of the isotropic phase at the expense of an erosion of the initially nematic phase. Furthermore, the nematic phase exhibits a collective rotational motion. The density difference between these two regions at different shear rates, allows us to determine the binodal line of the phase diagram. The results are in good agreement with experimental observations.

Phase transitions occurring in soft matter systems are signicantly affected by ow. Both the nature and location of the phase transition lines are changed due to the applied ow. The challenge is to nd the parameters that determine the non-equilibrium steady states under ow conditions. Colloidal-rod suspensions constitute a particularly interesting system to study the effect of ow on their phase behavior, since rod orientation is strongly coupled to the shear eld. Rods in the isotropic (I) phase align with the ow and become paranematic (P). This suggests that the transition to the nematic (N) phase, where rods have orientational order, is facilitated by shear. On the other hand, rods in the nematic phase undergo a collective tumbling motion in the presence of shear ow. The question that then arises is how these two effects will affect I-N detailed understanding of the ow behavior of a model system of attractive colloidal rods is useful for industrial applications where shear alignment of elongated objects, such as carbon nanotubes, wormlike micelles, and polymers, play a role. The non-equilibrium phase diagrams of attractive colloidal rods in shear ow have been investigated by mesoscale hydrodynamic simulations and compared to small-angle light-scattering (SALS) and rheology experiments [1, 2]. Earlier rheology experiments [3] have studied the non-equilibrium binodal of fd-virus dispersions under shear ow conditions for a single,

FIG. 1: Snapshots of the simulation box with = 3.5, (a) at

equilibrium, and (b) in a tumbling event at = 0.003. Col ors in (a) and (b) are coding the rod orientation: horizontal is red, vertical is green and perpendicular to plane of view is blue. Red arrows in (b) denote ow direction. (c) Time evolution of the normalized density /0 , and (d) of the orientational order parameter Sx , along the gradient direction.

xed strength of attraction. This study showed that the P-N transition changes on applying ow. The simulations allow for a microscopic understanding of the behavior of coexisting phases and their interface under shear, including the possible role of collective tumbling motion of rods. The attractive rod-rod interactions are systematically varied, which affects the phase behavior, interfacial properties of coexisting phases as well as the tumbling behavior. Molecular dynamic simulations of rod-like colloids of aspect ratio 20 with attractive interactions (LennardJones potential with a minimum of , in units of the thermal energy kB T ), are combined with a mesoscopic description of the solvent known as multiparticle-collision dynamics (MPC). This hybrid approach has been shown to account for long-range

18

FIG. 2: Non-equilibrium phase diagram obtained from simulations for various values of the strength of attraction interaction, with shear rates normalized by the maximum shear versus the fraction of equilibrium nematic phase. The inset presents the unscaled data. The dashed lines are the conjectured master curve, which are identical in the actual study and in the experimental one [1].

hydrodynamic interactions between rods [4]. The simulated system is prepared in equilibrium with coexisting isotropic and nematic phases, where the director of the nematic phase is aligned parallel to the interface. A snapshot of I-N coexistence is shown in Fig. 1a. We determine the equilibrium phase diagram as a function of the strength of an attractive interaction between the colloidal particles. We nd a widening of the two-phase coexistence region, which is small for weak interactions strengths, and becomes very pronounced for stronger interactions. This result very nicely agrees with recent experimental results in equilibrium [5] in which attraction is induced by a variable amount of depleting polymer. Shear is then applied with the imposed ow direction parallel to the interface, (vx , vy , vz ) = (y, 0, 0), with the applied shear rate. Shear ow orients the particles in the initially isotropic phase. This generates free space around each rod and facilitates the transfer of rods from the nematic into the paranematic layer. This mechanism is responsible for the reduction of the density difference between the two phases. Shear ow also induces a rotational motion of individual rods. More interestingly, it leads to a collective rotation motion of large groups of rods in the nematic phase, as indicated in Fig. 1b. This behavior is characterized by both the local concentration and the local orientational order parameter Sx (y) 3 ux ux 1 /2, where ux is the compo nent of the unit vector connecting the end-points of a rod along the ow direction, and the overline indicates averaging over the vorticity and ow directions. The time dependence of the density and orientational order parameter Sx of rods as a function of the position y along the gradient direction is plotted in Fig. 1c,d. As can be seen from Fig. 1c, the nematic phase has a higher concentration than the isotropic phase, as expected. More importantly, Fig. 1d demonstrates the periodic tumbling motion of rods in the nematic phase. This is seen for all nematic domains in coexistence with paranematic regions and for all strengths of attractions studied.

Binodals are determined at times where the density of the paranematic state has reached a stationary value and are plotted in Fig. 2 for different attractions. The inset in Fig. 2 shows that an increasing attraction between rods broadens the coexistence region and leads to an increase of max . The the coexis tence region widening is the same effect as observed in equilibrium. The factor max can be identied with the maximum of the binodal. Simulations for shear rates just above max indicate in fact that the sys tem evolves into an homogeneous state. In Fig. 2, the concentration is expressed in terms of nem , i. e. the proportion of the nematic phase in the equilibrium state. The shear rate is scaled by max . With this normalization, all data points fall onto a master curve. Precisely the same master curve was also extracted from experimental results. From this striking result we conclude that the effect of attractive interactions on the non-equilibrium phase diagram is reduced to two parameters, max and the difference in packing fractions between the isotropic and nematic phase in equilibrium. The collective rotational motion induced by shear has been observed only in the coexisting nematic phase. This is in contrast to the paranematic phase that remains ow-aligned without any collective motion. The interface between the coexisting tumbling and ow aligned states is therefore highly dynamic. The periodic motion of the nematic director during tumbling and kayaking is characterized by a time between subsequent ow-aligned states. Although this time is not uniquely dened, a clear trend can be observed. We nd that decreases linearly with the inverse shear rate, as observed previously well inside the nematic region. Furthermore, we observe that the tumbling time increases with increasing interaction strength. We believe that this is due to the higher packing fraction of rods in the nematic phase. This implies that the distance between rods is smaller, and therefore the hydrodynamic friction for their motion parallel to each other is larger. We want to emphasize that no nematic tumbling states have been found in the homogeneously nematic phase obtained for rates above the maximum of the binodal. This agrees with the experimentally determined tumbling-to-aligning phase separation line that appears at high concentrations and high shear rates and ends at the maximum of the binodal.

[1] M. Ripoll, P. Holmqvist, R. G. Winkler, G. Gompper, J. K. G. Dhont, and M. P. Lettinga. Phys. Rev. Lett. 101, 168302 (2008). [2] M. Ripoll, R. G. Winkler, K. Mussawisade, and G. Gompper, J. Phys.: Cond. Matt. 20, 404208 (2008). [3] M. P. Lettinga and J. K. G. Dhont. J. Phys.: Cond. Matt. 16, S3929 (2004). [4] R. G. Winkler, K. Mussawisade, M. Ripoll, and G. Gompper, J. Phys.: Cond. Matt. 16, S3941 (2004). [5] Z. Dogic, K. R. Purdy, E. Grelet, M. Adams, and S. Fraden, Phys. Rev. E 69, 051702 (2004).

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Cooperation of sperm in two dimensions: synchronization and aggregation through hydrodynamic interactions
Y. Yang, E. Elgeti, G. Gompper
IFF-2: Theoretical Soft-Matter and Biophysics

Sperm swimming at low Reynolds number have strong hydrodynamic interactions when their concentration is high in vivo or near substrates in vitro. The beating tails not only propel the sperm through a uid, but also create ow elds through which sperm interact with each other. We study the hydrodynamic interaction and cooperation of sperm embedded in a twodimensional uid. Two effects of hydrodynamic interaction are found, synchronization and attraction. With these hydrodynamic effects, a multi-sperm system shows swarm behavior with a power-law dependence of the average cluster size on the width of the distribution of beating frequencies. Sperm motility is important for the reproduction of animals. Before they nd the ova, sperm have to overcome many obstacles in their way. A healthy mature sperm of a higher animal species usually has a agellar tail, which beats in a roughly sinusoidal pattern and generates forces that drive uid motion. The snake-like motion of the tail propels the sperm through a uid medium very efciently. In nature, the local density of sperm is sometimes extremely high. For example, in mammalian reproduction, the average number of sperm per ejaculate is tens to hundreds of millions, so that the average distance between sperm is on the scale of ten micrometers, comparable to the length of their agellum. On this scale, the hydrodynamic interaction and volume exclusion are not negligible. In experiments with rodent sperm at high densities [1], motile clusters consisting of hundreds of cells have been found, which show much stronger thrust forces than a single sperm. The sperm seem to take advantage of strong interactions with neighbor cells of the same species to win the fertilization competition. The higher animal sperm typically have tails with a length of several tens of micrometers. At this length scale, viscous forces dominate over inertial forces. Thus, the swimming motion of a sperm corresponds to the regime of low Reynolds number. We describe the motion of the surrounding uid by using a particlebased mesoscopic simulation method called multiparticle collision dynamics (MPC) [2, 3]. This simulation method has been shown to capture the hydrodynamics and ow behavior of complex uids over a wide range of Reynolds numbers very well [3]. In

particular, it describes the helical motion of swimming sperm in three dimensions [4]. Here, we construct a coarse-grained sperm model in two dimensions. The sperm model consists of particles connected by stiff springs, which form a circular head and a lament-like tail, see Fig. 1. The beating motion is determined by a time-dependent, spontaneous curvature cs,tail (x, t) = c0,tail + A sin 2fs t + qx + s , which generates a propagating, sinusoidal wave along the tail. The wave number q = 4/L0 is chosen to mimic the tail shape of sea-urchin sperm, and L0 is the tail length. fs is the beating frequency of the s-th sperm. The constant c0,tail determines the average spontaneous curvature of the tail. s is the initial phase of the s-th sperm, and A is a constant related to the beating amplitude. We analyzed the hydrodynamic interaction between two sperm and the aggregation behavior of multisperm system. In our simulations, sperm clusters are always seen after the system has reached a stationary state. We concluded that hydrodynamic interaction and volume exclusion play important roles in the cluster formation of healthy and motile sperm. We also found that the average cluster size has a powerlaw dependance on the width of beat frequency distribution [5].

FIG. 1: Head-head distance dh of two cooperating sperm.

Simulation data are shown for xed phase difference (red, ). The interpolating (red) line is a linear t for 0.4 < < 1.5. The distance dh is also shown as a function of time t (top axis) in a simulation with a 0.5% difference in the beat frequencies of the two sperm (solid line).

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In two-sperm simulations, we found two effects of the hydrodynamic interaction, synchronization and attraction. When two sperm are close in space and swimming parallel, they synchronize their tail beats by adjusting their relative position. This synchronization" process is accomplished in a very short time. Then, the synchronized sperm have a tendency to get close and form a tight pair. This attraction" process takes much longer time then synchronization. Two sperm stay locked in phase and swim together until their phase difference becomes too large. This coordinating behavior decreases the total energy consumption of two sperm. Fig. 1 shows the linear relation between phase difference and headhead distance of a sperm pair. The cooperation of our sperm model fails when the phase difference > 1.5. With the knowledge of hydrodynamic interaction between two sperm, we study multi-sperm systems. Synchronization and attraction effects help the sperm to form large, motile clusters. We give each sperm random initial position and orientation. Considering that in real biological systems the beat frequency is not necessary the same for all sperm, we perform simulations with Gaussian-distributed beating frequencies. f = (f )2 1/2 / f denotes the width of the Gaussian frequency distribution. Here, (f )2 is the mean square deviation of the frequency distribution, and f = 1/120 is the average frequency.

FIG. 3: Dependence of the average stationary cluster

size, < nc >, on the width of the frequency distribution f . Data are shown for a 50-sperm system ( ) and a 25sperm system (). The lines indicate the power-law decays 0.201 0.196 < nc >= 2.12 f (upper) and < nc >= 1.55 f (lower).

size always increases for f = 0. For f > 0, however, sperm cells can leave a cluster after sufciently long time, since the phase difference to other cells in the cluster increases in time due to the different beat frequencies. At the same time, the cluster size can grow by collecting nearby free sperm or by merging with other clusters. Thus, there is a balance between cluster formation and break-up when f > 0. Once a system reaches a stationary state, the cluster-size distribution function obeys a power law. Similar power-law behaviors have been found in simulation studies of swarm behavior of self-propelled particles [6]. The average cluster size < nc > is plotted in Fig. 3 as a function of the width f of the frequency distribution. Generally, it decreases with decreasing sperm density or increasing f . Figure 3 also shows a power law decay,
< nc > f

(1)

with = 0.200.01. The negative exponent indicates that the cluster size diverges when f 0.

FIG. 2: A snapshot from simulation of 50 sperm with widths

f = 0.9% of a Gaussian distribution of beating frequencies. The red ellipses indicate large sperm clusters. The black frames show the simulation boxes. Note that we employ periodic boundary conditions.

[1] S. Immler, H. D. M. Moore, W. G. Breed and T. R. Birkhead, PLos One 2, e170 (2007). [2] A. Malevanets and R. Kapral, J. Chem. Phys. 110, 8605 (1999). [3] G. Gompper, T. Ihle, D. M. Kroll, and R. G. Winkler, Adv. Polym. Sci. 221, 1 (2009). [4] J. Elgeti and G. Gompper, NIC proceedings, Vol. 39 of NIC series, edited by G. Mnster, D. Wolf, and M. Kremer, pp. 53-61 (2008). [5] Y. Yang, J. Elgeti, and G. Gompper, Phys. Rev. E 78, 061903 (2008). [6] C. Huepe and M. Aldana, Phys. Rev. Lett. 92, 168701 (2004).

The motile sperm embedded in a two-dimensional uid aggregate through hydrodynamic interactions and volume exclusion. Fig. 2 shows a snapshot of a 50-sperm system with f = 0.9%. Large clusters of synchronized and tightly packed sperm are formed. If f = 0, once a cluster has formed, it does not disintegrate without a strong external force. A possible way of break-up is by bumping head-on into another cluster. As the cluster size increases, the possibility to bump into another cluster decreases, thus the rate of break-up decreases. The average cluster

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Twist grain boundaries in cubic surfactant phases

M. Belushkin, G. Gompper
IFF-2: Theoretical Soft-Matter and Biophysics

Cubic surfactant phases are mesoscale liquidcrystalline structures in which the surfactant monolayer separating the oil-rich and waterrich domains often has a triply-periodic minimalsurface geometry where the mean curvature H vanishes on the whole surface. The lattice constant which corresponds to the dimensions of the fundamental building block - the unit cell is large, of the order of 10 nm. During the nucleation of a cubic phase, many classes of defects arise. We have investigated twist grain boundaries in the lamellar, gyroid, diamond and Schwarz P phases. The structure of the monolayer in the grain boundaries is found to be very close to a minimal-surface geometry. The interfacial free energy per unit area is found to be very small, therefore the density of grain boundaries should be high in these surfactant phases. Amphiphilic molecules added to an immiscible oilwater system self-assemble into a large variety of structures. Phases with cubic symmetry often feature a triply-periodic minimal surface (TPMS) conguration of the surfactant monolayer. Examples of such phases include the gyroid G, diamond D, Schwarz P , Schoen I W P , F RD, Neovius C(P ) and others, which are encountered in physical systems. Properties of cubic surfactant phases have been a subject of extensive theoretical and experimental interest, with applications in biological systems, as templates for mesoporous systems and for the crystallization of membrane proteins. In amphiphilic systems many kinds of interfaces occur: between two ordered phases, between ordered and disordered phases, and between two grains of the same ordered phase which differ by their spatial orientation. Experiments on block copolymer systems have revealed the structure of many interfaces. For example, twist grain boundaries in the lamellar phase are well described by Scherks minimal surfaces at large twist angles and are helicoid-shaped at small twist angles [1, 2], whilst tilt grain boundaries in the lamellar phase have been shown to be omegashaped at large tilt angles and chevron-shaped at small tilt angles [3]. We report here on the investigation of twist grain boundaries in cubic surfactant phases. The calculations are based on a Ginzburg-Landau theory

of ternary amphiphilic systems [4, 5] with a single scalar order parameter (r) which describes the local oil-water concentration difference. The geometrical properties of the grain boundaries are evaluated on the isosurface (r) 0 which denes the position of the surfactant monolayer. The interfacial free energy per unit area of a grain boundary depends on the angles with respect to the crystalline axes. It is determined using the Ginzburg-Landau theory and a complementary geometrical approach based on the Canham-Helfrich curvature Hamiltonian. The calculations are performed on a discrete threedimensional real-space lattice ijk . Initially, half of each calculation box is lled with a phase rotated by an angle /2 and the other half with a phase rotated by an angle +/2. The free energy is minimized using a method based on the gradient descent algorithm [6].

FIG. 1: Conguration of the surfactant monolayer for the

lamellar phase at a twist angle of 53o (left) and the gyroid phase at a twist angle of 90o (right). The grain boundaries are located in the middle and at the top/bottom of each box.

Congurations of the surfactant monolayer in the full simulation boxes of the lamellar phase at a twist angle of 53o and the gyroid phase at a twist angle of 90o are shown on Fig. 1.

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The locations of the grain boundaries are determined quantitatively by relating the inital and nal simulation boxes [6]. In the lamellar, gyroid and diamond phases the thickness of the grain-boundary regions is about one unit cell of the bulk-phase regions, thus grain boundaries preserve the typical length scales of the bulk phases. The bulk-phase regions of the Schwarz P phase are greatly affected by the presence of grain boundaries. The surfactant monolayers in grain-boundary regions of the lamellar (top), gyroid (middle) and diamond (bottom) phases are shown on Fig. 2 for twist angles of 53o (left) and 90o (right).

Gaussian-curvature distributions (Fig. 3) show that [6] the Gaussian curvature distributions in the bulkphase regions are consistent with the exact results obtained from the Weierstrass representation in the L , G and D phases the geometry of the grain boundaries is signicantly different from the geometry of the bulk phases Excess free energy per unit area of the grain boundaries determined from Ginzburg-Landau theory as a function of the twist angle is shown on Fig. 4. Geometrical approaches based on the Canham-Helfrich curvature energy Hamiltonian yield similar results [6]. The excess free energy of the grain boundaries exhibits a non-monotonous dependence on the twist angle , and the negative values for the P phase show it to be unstable with respect to the nucleation of grain boundaries at the investigated point in the phase diagram.

FIG. 2: Twist grain boundary geometries for the lamellar

(top), gyroid (middle) and diamond (bottom) phases for twist angles of 53o (left) and 90o (right).

Squared-mean and Gaussian curvature distributions are calculated for each phase and twist angle. Squared-mean-curvature distributions show that the mean curvature of the surfactant monolayer essentially vanishes both in the bulk-phase and grainboundary regions, therefore the congurations of the surfactant monolayer are good approximations of minimal surfaces [6].

FIG. 4: Grain-boundary excess free energy per unit area

as a function of the twist angle for the lamellar, gyroid G, diamond D and Schwarz P surfactant phases as extracted from Ginzburg-Landau theory.

The free energy of the grain boundaries is very small. The difference in the grain-boundary free energy and the free energy of bulk-phase regions of equal volume is of the order of 1% at the maxima of Fig. 4. Therefore, the density of grain boundaries should be high in these surfactant phases.

[1] S. Gido et al., Macromolecules 26, 4506 (1993) [2] H. Jinnai et al., Macromolecules 39, 5815 (2006) [3] Y. Cohen et al., Macromolecules 33, 6502 (2000) [4] G. Gompper, M. Schick, Phys. Rev. Lett. 65, 1116 (1990) [5] U. S. Schwarz, G. Gompper, Phys. Rev. E 59, 5528 (1999) [6] M. Belushkin, G. Gompper, J. Chem. Phys. 130, 134712 (2009)

FIG. 3: Gaussian-curvature distribution for the G phase at

a twist angle = 27.8o . The blue solid line corresponds to the exact result obtained from the Weierstrass representation. The black vertically-shaded and red slant-shaded histograms correspond to bulk and grain-boundary regions, respectively.

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Clustering and alignment of red blood cells in microcapillaries

J. L. McWhirter, H. Noguchi, G. Gompper
IFF-2: Theoretical Soft-Matter and Biophysics

The shapes, aggregation, and alignment of red blood cells (RBCs) in cylindrical capillary ow is investigated by mesoscopic hydrodynamic simulations. We study the collective ow behavior of many RBCs, where the capillary diameter is comparable to the diameter of the RBCs. Two essential control parameters in our study are the volume fraction of RBCs (the tube hematocrit, HT ), and the ow velocity of the RBC suspension. At very high ow velocities and very low HT , the parachute-shaped RBCs can be found in clusters that are stabilized by the hydrodynamic solvent ows. At high HT , the RBCs can exhibit one disordered phase and two distinct ordered phases depending on HT and the ow velocity. Thermal uctuations, included in the simulation method, coupled to hydrodynamic ows are important contributors to the RBC morphology.

FIG. 1: Sequential simulation snapshots of six RBCs in

a dilute blood suspension (L = 3.3 or HT = 0.084 = ves 2 nves Vves /Lz Rcap = 0.28/L ) at v0 = 7.7 and nves ves = 6. The full length of the cylindrical simulation tube is Lz . The critical ow velocity associated with the shape transition from a discocyte at low velocities to a parachute at higher velocities is vc 5. Top panel shows a 6 RBC cluster while the bottom two show the break-up of this cluster at a later time.

In the absence of ow, human RBCs have a biconcave-disc (discocyte) shape whose maximum diameter and thickness are 7.6 m and 2 m with constant area and volume, Vves . The RBC membrane consists of a lipid bilayer supported by an attached spectrin network, which acts as a cytoskeleton and is responsible for the shear elasticity of the membrane. The bilayers resistance to a bend is controlled by a curvature energy with a bending rigidity, , and the spectrins resistance to a shear strain is characterized by a shear modulus, . At thermal equilibrium, the discocyte shape of a RBC can be predicted theoretically by minimizing the membranes bending and stretching energy subject to a xed surface area and volume. However, under owing, nonequilibrium conditions, the shape adopted by the RBCs is determined by the competition between these mechanical properties and the external hydrodynamic ow forces arising from the blood plasma (the solvent) suspending the RBCs. The spectrin network enables an RBC to remain intact while changing its shape in blood ow through narrow capillaries with diameters of 0.2 m to 10 m. At high dilution in fast blood ows through narrow capillaries, optical microscopy of micro vessels [1] have shown that individual RBCs can adopt a parachute shape; the faster ow in the center of the capillary and the slower ow near the walls in-

duces this shape change. The RBC deformability is reduced in blood related diseases, such as diabetes mellitus and sickle cell anemia; with these diseases the resistance to ow is relatively high and the heart must work harder to produce the higher pumping pressures needed to ensure normal blood ow. Therefore, the deformability of a RBC is important for the regulation of oxygen delivery. We have already studied the shape changes of a single, isolated RBC in a blood ow at high dilution [2] using a mesoscopic simulation technique which combines two methods. The solvent hydrodynamics is described by an explicit, particle-based dynamics called Multi-particle Collision Dynamics (MPC). The RBC membrane is treated by a discretized mechanical model of a two-dimensional elastic membrane parametrized by and , that is, a dynamically triangulated surface model [3]. Our studies showed that the shape change of an RBC from a discocyte to a parachute occurs at a critical ow velocity which depends on the material parameters and [2]. As the RBC became more rigid, a faster the blood ow was needed to achieve this shape transition. This transition was accompanied by a sudden drop in the pressure needed to drive the ow at a given velocity. The main focus of the present work is to study the collective ow behavior of many interacting RBCs at very low and high HT [4, 5]. We consider a number nves of vesicles in capillary tube segments of length Lz and radius Rcap with periodic boundary conditions in the ow direction, Z. Here L = Lz /nves Rcap ves

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FIG. 2:

Simulation snapshots of nves = 6 RBCs: (a) Disordered-discocyte phase for L = 0.875 and v0 = 2.5; ves (b) Aligned-parachute phase for L = 0.875 and v0 = 10; ves and (c) Zigzag-slipper phase for L = 0.75 and v0 = 10. ves
and v0 v0 /Rcap , where the characteristic shape 3 relaxation time is = 0 Rcap /. A gravitation force mg is used to generate ow along the Z axis where 2 v0 = mns gRcap /80 and 0 is the viscosity of the suspending solvent.

FIG. 3: (a) Phase behavior as a function of average vesicle

distance L and mean ow velocity v0 , for nves = 6. The ves hematocrit varies between HT = 0.22 and HT = 0.45, since HT = 0.28/L . Symbols represent the disorderedves discocyte ( ), aligned-parachute ( ), and zigzag-slipper () phases, respectively. (b) Pressure drop Pdrp per vesicle for the aligned-parachute phase (simulations with nves = 1) and the zigzag-slipper phase (simulations with nves = 6) at the same volume fraction (L = 0.75, corresponding to ves HT = 0.37).

At very low HT , parachute shaped RBCs form sta ble clusters at ow velocities v0 much higher than the critical ow velocity, vc , associated with the discocyte to parachute transition. There are two reasons for the cluster formation. First, a single RBC separated from a neighbouring cluster of RBCs is more deformed by the ow than its neighbours, bending closer to the capillary axis or center where the ow is fastest; therefore, this single RBC approaches the cluster, forming a larger cluster. Second, the effective hydrodynamic ow mediated attractions between the RBCs in a cluster stabilize the cluster, increas ing its lifetime. As v0 approaches vc , uctuations in the RBC shape increase, leading to cluster break-up events as shown in Fig. 1. At high HT , three distinct RBC phases exist (Fig. 2); one is disordered, the disordered-discocyte (D) phase, while the remaining two are ordered, the aligned-parachute (P) and zig-zag slipper (S) phases (Fig. 3). In the D phase at L 0.85 and low ves v0 , the RBCs appear as discocytes with random orientations and no signicant long-range spatial cor relations. The P phase was expected at higher v0 based on the simulations examining the shape transition to a parachute of a single, isolated RBC under ow [2]. However, given these earlier simulations, the S phase occurred unexpectedly at smaller L . Here, slipper-shaped RBCs form two regular, ves interdigitated parallel rows. Curiously, the S phase produces a larger pressure drop Pdrp , or resistance to ow, than the periodic P phase under equivalent conditions, as shown in Fig. 3(b). An alignedparachute conformation at L 0.85 is destabives

lized by the thermal uctuations that are incorporated into the mesoscopic simulation method; these uctuations have the effect of moving an RBC off the capillary axis to regions of slower ow. In other theoretical approaches, thermal uctuations are not incorporated, or their incorporation is not as simple as in MPC. In summary, we have shown that at very low RBC volume fractions the RBC deformability implies a owinduced cluster formation at high blood ow velocities. In addition, at high volume fractions, the collective behavior of several RBCs determines their owinduced morphology and resistance to ow. Future studies will examine the dependence of ow properties on channel geometry, polydispersity of the RBC suspension, and the introduction of more rigid, spherical white blood cells.

[1] Y. Suzuki, N. Tateishi, M. Soutani, and N. Maeda, Microcirculation 3, 49 (1996). [2] H. Noguchi and G. Gompper, Proc. Natl. Acad. Sci. 102, 14159 (2005). [3] H. Noguchi and G. Gompper, Phys. Rev. E 72, 011901 (2005). [4] J.L. McWhirter, H. Noguchi, and G. Gompper, Proc. Natl. Acad. Sci. 106, 6039 (2009). [5] P. Gaehtgens, C. Dhrssen, K.H. Albrecht, Blood u Cells 6, 799 (1980).

25

26

IFF-5: Neutron Scattering

Director: Prof. Dieter Richter

The Institute for Neutron Scattering is concerned with neutron research placing major emphasis on soft condensed matter, i.e. materials that react strongly to weak forces. Neutron scattering is a valuable tool for these systems because it reveals structure and dynamics of Soft Matter on the relevant length- and timescales. A major part of the Soft Matter studies is done on polymers. Apart from their structure, we are interested in the dynamics of polymers in melts and solutions (e.g. gels, rubbery networks, aggregates). These polymers often have a complex architecture (copolymers, star-polymers etc.) to tailor them for industrial applications. Another field of interest are complex liquids such as microemulsions or colloid systems.

Biological materials (e.g. proteins) are studied concerning their structure and dynamics. The institute has modern chemical laboratories for the synthesis, characterisation, and modification of Soft Matter. In order to complement neutron scattering experiments several ancillary techniques are used in the institute: rheology, light scattering, calorimetry, x-ray scattering, impedance spectroscopy, and computer simulation. The Institute for Neutron Scattering is partner in the Jlich Centre for Neutron Science JCNS. In this position it operates several neutron scattering instruments at the research reactor FRM II in Munich, at the Institut Laue-Langevin in Grenoble, and at the Spallation Neutron Source in Oak Ridge, USA. These instruments are available to guest researchers on request. Another focus of research is the development of neutron instrumentation for research reactors and future spallation sources worldwide.

27

Synthesis of Polyalkylene Oxide Homo- and Block Copolymers

J. Allgaier
IFF-5: Neutron Scattering

The anionic ring-opening polymerization of alkylene oxides like propylene or butylene oxide is accompanied by strong side reactions. This leads to large amounts of byproducts. In this work the anionic polymerization of hydrophobic alkylene oxides was investigated at different temperatures, solvents and initiating systems [1]. For polymers synthesized above room temperature significant amounts of byproducts were found. With the help of crown ethers the polymerization temperature could be reduced to -23C. This measure allowed eliminating by-products almost completely in the synthesis of poly(1,2-butylene oxide), poly(1,2-hexylene oxide), and poly(1,2octylene oxide). Furthermore the method was employed to synthesize amphiphilic block copolymers of these polyalkylene oxides with polyethylene oxide as the hydrophilic moiety. The new synthetic method is generally of interest due to the rheological and dielectric properties of the polymers and the possibility to tune amphiphilicity of the amphiphilic block copolymers. In contrast to the limited availability of the higher members, the polyalkylene oxide family offers interesting possibilities with respect to their rheological properties due to the systematic variability of the polymer chain diameters. Because of the dipole moment along the chain PAO represents an alternative to the widely used polyisoprene for dielectric measurements. In addition, by combining PEO with the other PAO, the amphiphilicity of the resulting block copolymers can be varied systematically. In contrast to most other block copolymers the hydrophobic moieties in the PAO block copolymers are soluble in a large variety of oils, making them ideal candidates as additives in microemulsion systems. The lowest members of the polyalkylene oxide (PAO) family, polyethylene oxide (PEO) and polypropylene oxide (PPO), are known for a long time. The most widely employed technique for the polymerization of alkylene oxides is anionic ring-opening polymerization using sodium or potassium alcoholates as initiators. This technique allows to polymerize ethylene oxide (EO) basically free of side reactions. For the polymerization of propylene oxide (PO) strong side reactions are present. The reason for the side reactions is the relatively high acidity of the methyl protons of PO leading to

different types of termination and chain transfer reactions. As a result high and low molecular weight by-products are formed [2, 3]. In the older literature, there are reports dealing with the synthesis of the higher and more hydrophobic PAO than PPO. Poly(1,2-butylene oxide) (PBO), poly(1,2-hexylene oxide) (PHO), poly(1,2octylene oxide) (POO) and even higher PAO were obtained by polymerizing the corresponding monomers with zinc organic catalysts [4-6]. This technique, however, yields polymers with broad MWD and does not allow producing block copolymers. Figure 1 illustrates the chemical structures of some polyalkylene oxides and the corresponding monomers.
monomer O H2C CHR polymer

( CH2

CH

R=H R=CH3 R=CH2-CH3 R=(CH2)3-CH3 R=(CH2)5-CH3

ethylene oxide propylene oxide 1-butylene oxide 1-hexylene oxide 1-octylene oxide

R polyethylene oxide (PEO) polypropylene oxide (PPO) polybutylene oxide (PBO) polyhexylene oxide (PHO) polyoctylene oxide (POO)

FIG. 1: Structures of different alkylene oxide monomers and polymers.

In a first series of experiments butene oxide was polymerized at different temperatures and using different solvents and initiators. The target molecular weight, defined by the amounts of monomer and initiator introduced into the reactor, was 15,000. The SEC trace of sample PBO-1 is given in Figure 2. In this experiment potassium tert.-butanolate (KOt-Bu) was used as initiator at 80oC and toluene was chosen as an inert solvent. Beside the main peak the signal contains a low molecular weight tailing and a high molecular weight shoulder at higher and lower elution volumes. In agreement with this result the molecular weight distribution is elevated and the measured molecular weight is about 25% smaller than the calculated molecular weight, obtained from the amounts of initiator and polymerized

28

monomer. In further experiments other alkali metal initiators and more polar aprotic solvents were tested in order to reduce the polymerization temperature without reducing reactivity and extending the polymerization time too much. This measure was successful insofar the by-product content could be reduced. However, at 40oC still significant amounts of by-product were present and at lower temperatures polymerization was extremely slow.

PBO-PEO

PBO

P BO-1

P BO-15

34

elution volume

40 4 0

FIG. 3: SEC refractive index signals of a PBO-PEO block copolymer and the corresponding PBO homopolymer with block molecular weights of approximately 10,000.

35

40 elution volume

35

40 elution volume

FIG. 2: SEC refractive index signals of PBO samples, polymerized without crown ether at 80C in toluene (PBO-1) and with crown ether at -20C in toluene (PBO15).

Therefore a different strategy was chosen based on crown ethers. These additives are strong complexing agents for alkali metal ions. Especially in less polar solvents they increase the degree of ion-pair separation. In case of alkylene oxide polymerization this leads to increasing reactivities and polymerization rates. In our case it was possible to reduce the polymerization temperature considerably below room temperature. Because of the reduced level of byproducts already achieved without crown ethers, the target molecular weight was increased from 15,000 to 50,000. This measure generally increases the relative fraction of by-products and helps to get a clearer picture about improvements of the product quality. Again different alkali metal initiators in combination with different crown ethers were tested. It turned out that KOt-Bu together with the crown ether 18C6 was the best combination. Using this system the polymerization temperature could be reduced o below -20 C and within experimental error no byproduct could be detected (see Figure 2, sample PBO-15). The more hydrophobic monomers hexene oxide and octene oxide could be polymerized under similar conditions as butene oxide. However it was necessary to purify those monomers by careful fractional distillation due to the lower purity of the commercial raw material compared to butene oxide. With this additional measure molecular weight of 50,000 to 100,000 could be obtained without significant by-product content and low molecular weight distributions.

A-B block copolymers containing PBO, PHO, and POO as hydrophobic blocks and PEO as hydrophilic block were produced using the polymerization techniques described before. This technique allows to synthesize the block copolymers by successively polymerizing the hydrophobic alkylene oxide and EO in a one-potreaction. Within experimental error the calculated compositions and the measured ones were similar. The small values of Mw/Mn indicated narrowly distributed polymers. As an example the SEC trace of a PBO-PEO block copolymer having block molecular weights of 10,000 is given in Figure 3. It shows a symmetrical signal. At higher elution times there is no hint for homopolymer. Even block copolymers having block molecular weights of 50,000 were basically free of homopolymer. This again underlines that the new low-temperature method using crown ethers yields model polymers of high structural quality.

[1] Allgaier, J.; Wilbold, S.; Chang, T. Macromolecules, 2007, 40, 518. [2] Bailey, F. E., Koleske, J. V. In Alkylene oxides and their polymers, Surfactant Science Series; Schick , M. J., Frederick, M. F., Eds.; Marcel Dekker: New York, 1991; Vol. 35, Chapter 4. [3] Whitmarsh R. H. In Nonionic Surfactants Polyoxyalkylene Block Copolymers, Surfactant Science Series; Nace, V. M., Ed.; Marcel Dekker: New York, 1996; Vol. 60, Chapter 1. [4] Lal, J. J. Polym. Sci. A-1 1966, 4 . [5] Lal, J. J. Polym. Sci. B 1967, 5, 793. [6] Booth, C.; Orme, R. Polymer 1970, 11, 626.

29

Protein Domain Motions observed in Space and Time by NSE

Ralf Biehl1, Michael Monkenbusch1, Bernd Hoffmann2, Peter Falus3, Sylvain Prvost4, Rudolf Merkel2, Dieter Richter1
1 2

IFF-5: Neutron Scattering IBN-4: Biomechanics 3 Institut Laue Langevin, Grenoble, France 4 Hahn Meitner Institut, Berlin, Germany
To bind a cofactor to the active center of a protein often a structural change is mandatory. This induced fit includes small changes of single bonds but also large domain motions to prepare the active atomic configuration. Neutron spin echo spectroscopy is used to directly observe the domain dynamics of the protein alcohol dehydrogenase [1]. The collective motion of domains as revealed by their coherent formfactor relates to the cleft opening dynamics between the binding and the catalytic domains enabling binding and release of the cofactor to the active center. Proteins are the molecular machinery of life. As nanomachines of metabolism, they are in every cell of our body tirelessly active, transport, synthesize, divide and transform substances. Many transformation processes are underway in characteristic shaped bags, in which only certain substances fit, as a key fits to the lock. The shape of these pockets is determined by the sequence and three-dimensional arrangement of amino acids. Sometimes the binding of the cofactor or substrate is responsible for the formation of the right shape of the pocket. Through this "induced fit" the lock for a certain key is arranged to fit. There must be movements of atoms, amino acids or entire domains to bring the atoms in the right configuration to get active. This effect is so far mainly known from structural studies of crystallized proteins, which represent snapshots of different configurations. Internal movements could also be observed by fluorescent labelling of two points on a protein with molecular biological methods to track distance changes [2]. Further information is attainable only by computer simulations. One of the most studied proteins and one of the key enzymes is Alcohol Dehydrogenase (ADH, Figure 1). It produces ethanol in yeast or converts it back to acetaldehyde in the human liver. A functional important molecule (cofactor) Nicotinamide Adenine Dinucleotide (NAD) and a substrate molecule (ethanol or acetaldehyde) bind to the binding domain in a cleft between active and binding domain. The active domain, with a catalytic Zinc atom in the active centre, opens and closes the cleft by thermal movements. The closing places the active centre in the right position for the transfer of a hydrogen atom between the substrate and cofactor molecule, and another hydrogen atom is released. Again opened, the cofactor and the product of the reaction will be released.

FIG. 1: Tetrameric Alcohol Dehydrogenase of yeast is build up from two dimers (green-blue and gray). The blue monomer is in the closed conformation with a NAD cofactor (orange) and an ethanol (red) in the cleft between binding domain and the active domain. In the cleft between both domains of the open configuration (green monomer) the catalytic Zinc atom at the catalytic centre is visible. The second dimer (gray) is bound at the backside of the first dimer. The arrow indicates schematically the movement of the active domain here without a bound cofactor.

Neutron scattering provides the possibility to investigate the timescale of these large scale movements in biomolecules. It provides information on the location and movement of atoms, without destroying the sample. The measurements were done in dilute D2O buffer solutions, close to the natural environment of the protein. D2O offers the possibility to focus on the protein scattering, because the D2O is only a weak scatterer for neutrons compared to H2O or the protons in the protein. Scattering at the atomic nuclei of the protein will produce a characteristic interference pattern.

30

Movements of the atoms during the scattering process change the speed of the neutrons in the scattered beam. The change in speed for the scattered neutrons is very small. In order to detect it, NSE spectroscopy uses the Larmor precession of the neutron spins in a magnetic field as a stopwatch. The result is a length scale dependent flexibility, which differs from the expected motion of a rigid protein, only showing translation and rotation movements of diffusion. These length scale variations reflect the pattern of the movements due to the underlying dynamics.

these motions as small variation of the rigid protein structure enables the calculation of the change in the effective diffusion due to overdamped normal mode motions. The change in the effective diffusion of the most prominent normal modes 7 and 11 is shown in Figure 3. Other modes show a smaller contribution. Applying the method to the protein with and without cofactor shows the influence of the cofactor onto these characteristic motional patterns. The peak structure is comparable to the measured peak in figure 2 and shows also the effect of a shift due to the bound cofactor.

FIG. 2: a, Single tetramer diffusion coefficient D0eff after corrections with and without bound cofactor. The black solid line represents the calculated effective diffusion coefficients for the ADH crystal structure, including translational and rotational diffusion of the stiff protein. At low q we observe only translational diffusion. The increase with the peak structure is due to rotational diffusion, which is only visible at larger q. b, Difference of the corrected diffusion coefficients and the calculated translational/rotational diffusion coefficient, reflecting the internal dynamics.

FIG. 3: Change of the effective diffusion due to movements along normal mode 7 and 11. Modes below 7 are trivial modes from translation and rotation. Without cofactor mode 7 is a mode which opens the cleft between catalytic domain and binding domain as indicated in figure 1. Mode 11 is a large amplitude mode described as torsion inside of the dimer along their main axis. The normal modes are numbered in sequence starting with the slowest timescale.

The NSE spectroscopy always measures the collective movement of all protein atoms, so in the evaluation the contribution of translational and rotational diffusion to the observed scattering has to be separated from the internal dynamics (Figure 2). After this we find a remarkable peak in the difference to the rigid body diffusion shown in Figure 2b. The internal motions of a protein are quite complex. One way to explore possible large scale motions in a protein is the analysis of elastic normal modes. This method uses a simplification, it assumes only elastic forces between a reduced number of atoms representing the main structure of the protein. The method is like building a simplified model of the protein with the same shape made from elastic rubber. By shaking the rubber model it is possible to observe large scale motions with weak parts as flexible hinges between more compact stiffer domains. Analysing

We found in our studies that the patterns of the movements observed are mainly due to the movement of the active domain relative to the binding domain at a timescale of 30 ns. Figure 1 illustrates schematically the movement of the active domain, leading to the opening of the gap according to normal mode 7. If the cofactor binds, it stiffens the protein, and the domain movement is significantly reduced. In ADH, we find an average amplitude of about 0.8 nm for the extent of the spatial movements between the two domains. Also we determined a spring constant of about 5 pN/nm for the stiffness of the cleft opening.

[1] Ralf Biehl, Bernd Hoffmann, Michael Monkenbusch, Peter Falus, Sylvain Prvost, Rudolf Merkel and Dieter Richter Phys. Rev. Lett. 101 (2008) 138102 [2] D. W. Pistona, G.-J. Kremers Trends in Biochemical Sciences, 32 (2007) 407

31

Unexpected power-law stress relaxation of entangled ring polymers

W. Pyckhout-Hintzen1 , D. Richter1 , D. Vlassopoulos2 , M. Rubinstein3
1 2 3

IFF-5: Neutron Scattering FORTH, Institute of Electronic Structure and Laser, Heraklion, Crete,Greece University of North Carolina, Department of Chemistry, Chapel Hill,USA

Linear and long-chain branched polymers relax by reptation processes out of the conning tube or via arm retraction respectively. Both mechanisms are intimately related to the presence of chain end material. It is of major interest how entangled ring polymers which lack this basic ingredient of chain ends proceed to relax stress. Self-similar dynamics yielding a power-law stress relaxation is reported on model ring polymers. Here, the importance of some linear impurities becomes evident. The combination of neutron scattering and rheology for rings is a promising tool therefore to unravel details of their dynamics and their difference with linear chains. The investigation of polymer dynamics is always closely related to the particular structure or even the architecture that the polymers adopt. Fig 1a represent a reptational-like motion through an obstacle eld of chains, which is clearly different from star uctuations in Fig 1b where only arm retraction modes can relax the stress. Rings in Fig 1c and 1d may occur in double-fold shape or even behave like latticeanimals respectively. Latter has a very strong similarity with a random cayley tree (Fig 1e) for which the relaxation time spectrum corresponds to long logarithmically-spaced time scales for each of the different chain sections. These follow a hierarchical scheme and relax as is well known from former works from the outside-inwards in a sequential way, leading to typical patterns in the complex relaxation modulus. For model-branched architectures, the number of generations is coupled to the number of loss peaks. It is, however, not obvious how a ring-like polymer will perform. Fig f depicts e.g. self-interpenetration and blockage of the rings. In the former considerations the inuence of linear contaminants which are the product of non-perfect linking chemistry or degradation of closed cycles to open linear chains, was not yet included. To verify theories which focus on the special structuredynamics associated with the odd architecture, it is a prerequisite to purify the rings. Recently, developments of liquid chromatography at the critical condition have shown to efciently separate rings and linear chains via the compensation of entropic size exclusion and enthalpic interactions with the pores. The polymers so obtained were analyzed by rheology and SANS in order to assure their unchanged,

original closed structure. The linear rheology for the puried rings is very compatible to the model of self-similar lattice animals of Fig 1(d,e). The associated exponentially decreasing relaxation modulus G(t) is then given as G(t) = GN 0 t e 2
5

exp

t ring

FIG. 1: Different correlations of structure with assumed

dynamics are summarized and explained in the text. The comparison of ring with branched polymers is made clear.

This is distinctly different in linear or branched polymers which show an extended entanglement plateau. This expression for the relaxation modulus already includes constraint-release effects for loop rearrangement as well as dynamic dilution and contains no further adjustable parameters. SANS measurements are shown in Fig 2. From dilute solution and in a melt of linear chains (not shown), unperturbed chain dimensions as well as the swelling of the ring by excluded-volume statistics could be

32

conrmed. The experimental slopes in Fig 2 are typical. Very good agreement in both size as well as the scattering amplitudes was obtained. The ring in good solvent is swollen by a factor of 1.5 which is in accordance with estimates of about 1.4 in literature. No traces of impurities of linear chain could be spotted from SANS.

We have forwarded in Fig 3 the molecular picture that linear chains must be bridged at this extremely low concentration by the rings to be so effective and therefore provide considerable increase in the melt viscosity. The entropically driven penetration of rings by linears leads then to a transient network. As both natural and synthetic polymers can be found in this severe cyclic architecture the present results are of both fundamental and practical signicance. Optimized microprocessing as well as rheology modications are thus within reach. Rings will be investigated further using adequately isotope-labeled systems to study differences in segmental dynamics and chain uctuations compared to their linear analogs. Also the question whether a plateau modulus shows up or power law relaxation persists if the rings get considerably larger will be investigated. Additionally blends with linear homopolymers will be addressed as well to be compared with recent advances in blends of linear with dendritic polymers. The techniques of interest are neutron spin echo (NSE) and small angle scattering (SANS) in quenched state which focus on the short times i.e. high-temperature motion and long times i.e. large-scale dynamics respectively. The present work was carried out in the framework of the Joint Programme of Activities of the SoftComp Network of Excellence (contract number NMP3-VT-2004-502235) granted under the FP6 by the European Commission.

FIG. 2: SANS experiments on dilute solutions under

and good solvent conditions show the characteristic scattering vector dependences. q2 is random-walk statistics on intermediate length scales whereas the q1.6 is prominent for excluded-volume interactions in swollen rings. The strong parasitic forward scattering at low q for the cyclohexane sample following q3.1 is due to density uctuations in the theta-state.

[1] M. Kapnistos, M. Lang, D. Vlassopoulos, W. Pyckhout-Hintzen, D. Richter, D. Cho, T. Chang, M. Rubinstein, Nature Materials, 7, 997-1002(2008) [2] M. Rubinstein, R. Colby, Polymer Physics, (Oxford University Press, 2003) [3] M. Rubinstein, Phys. Rev. Lett., 24, 3023-3026(1986)

FIG. 3: Linear chains are percolating through bridging by

the rings at low concentrations.

The effect of the linear chain on the dynamics of a ring could be quantied by deliberately mixing in the sample again known amounts of linear chains and remeasure the relaxation modulus. It shows that even at concentrations of 1/50th of the overlap concentration of the rings, the relaxation time spectrum gets a new long-lived component which grows substantially with concentration. The viscosity rises and a plateau shows up again. The understanding of this is important to relate former experiments in literature on rings which were not puried so extensively as here and therefore always showed a plateau modulus value.

33

Nucleation and Growth of CaCO3 Mediated by the Protein Ovalbumin

V. Pipich1, M.Balz2, S.E. Wolf2, W. Tremel2, D. Schwahn3
1 2

JCNS: Jlich Centre for Neutron Science Institute for Inorganic Chemistry, Johann Gutenberg-University, Mainz 3 IFF-5: Neutron Scattering

d/d [cm ]

Formation of CaCO3 mineralization in the presence of the egg-white protein ovalbumin was studied by time-resolved small-angle neutron scattering. A particular goal was to achieve a better insight into the early stages of mineralization. This is a relevant goal as the capability of mineral formation by living organism is not yet well understood. Starting from the amorphous phase several stages of mineralization, namely the crystalline polymorphs vaterite and aragonite, were observed. The formation and dissolution of 2+ amorphous CaCO3 is accompanied by Ca mediated unfolding and cross linking of about 50 protein monomers showing linear chain characteristic scattering. The protein complexes act as nucleation centers because of their enrichment by Ca2+ ions. Living organisms are capable of developing inorganic minerals with complex architectures to fulfill important biological functions, such as skeletal support or protection of soft tissues [1,2]. The mineral phase of such materials is intimately associated with organic macromolecules, such as proteins and polysaccharides. However, the role of these proteins in mineralization is not well understood. Small-angle neutron scattering (SANS) is a new technique in biomineralization which allows identification and structural analysis of biomineral composites by the method of contrast variation [2]. With the particular intention of a better insight into the early stages of mineralization, we performed studies of in-situ calcium carbonate formation mediated by the egg-white protein ovalbumin. Details of this work can be found in [3]. A characteristic result after 13 h mineralization by using the gas diffusion technique is shown in Figure 1. The scattering pattern is characterized by two power laws representing m large mineral particles and nm-sized proteins at small and large scattering vector Q, respectively. The Q-4 power law is a measure of the total mineral surface (Porods law: P4Q-4) whereas the Q-1 power law is caused from the linear chains of ovalbumin. So, the large difference in size of the mineral and protein allows a separate analysis of both constituents. Another important property of SANS is the large variation 2 range of scattering contrast of aqueous solutions by the exchange of H2O and D2O. The

symbol = ( S ) describes the difference of the coherent scattering length density of the mineral or protein and of the solvent S. The corresponding values are summarized in the Table together with representing the D2O content when the protein and the mineral polymorphs are matched in water, e.g. = 0 .

10 10 10 10

2.5mg/ml Ovalbumin

-4

-1

-1

-1

10

-3

10

-2 -1

10

-1

Q [ ] FIG. 1: Scattering pattern after 13 h mineralization in the presence of ovalbumin. The scattering from the mineral and protein are distinct by the two power laws

We performed several mineralization experiments in aqueous solution of different D2O content, in order to identify the mineral polymorphs. Figure 2 shows the time evolution of the amplitude P4 of four relevant contrasts. In all cases a peak becomes visible about 2.5 hours after initiation the mineralization. This scattering was identified from ACC particles as the intercept of P4 = 0 occurred at =0.550.05 (Table). So, the mineralization started with the formation of an amorphous polymorph, which after reaching a maximum volume fraction transformed to a crystalline polymorph. The pronounced minimum for the =0.74 D2O solvent after 4 h, and the smallest scattering of the =0.82 sample after 5 h provide the interpretation that CaCO3 follows a polymorph sequence of amorphous vaterite aragonite (Table). The scattering patterns in Figure 3 represent the contribution from the protein during mineralization (large Q part in Figure 1). During the first 1.5 h the scattering of the protein is best described by the

34

50 40 30
H2O D2O

0.30 0.25 0.20 d/d [cm ]

-1

-1

10 10 10 10

-2

-1

P4 [10 cm ]

-4

20 10 0 5 4 3 2 1 0 0
ACC Vaterite

-1

-1

0.15 0.10 0.05 0.00 0 1 2 3

-2

10

-3

10

-2

10

-1

-10

74% 82%

4
-2

5
-1

Q [10 ]
FIG. 3: Protein scattering during mineralization ( before start, after start of mineralization at 7 15 min , 90 min, and nearly 13h). A gradual aggregation of the proteins is observed. The inset shows ovalbumin in 0.1 M CaCl2 aqueous D2O solution. The enlarged scattering is caused by cross linking of about 52 proteins to an object with the conformation of a Gaussian linear chain -2 -1 as seen by the Q and Q power laws (Radius of gyration 600).

10

12

Time [h]
FIG. 2: Porod amplitude versus mineralization for different scattering contrasts. A peak from the amorphous polymorph (ACC) became visible at 3h. After 4h the minimum of the 0.74 D2O solution identifies the vaterite polymorph whereas aragonite appears as the stable phase within 12 h.

[10 cm ] Calcite Aragonite Vaterite Amorphous Ovalbumin Water 4.69 5.10 4.49 3.29 1.64+1.61 -0.561+6.95

10

-2

0.76 0.81 0.74 0.55 0.41 ---

seen from the reduction of the amplitude P4 in Figure 2. In a separate experiment of ovalbumin in 0.1 M CaCl2 aqueous solution we determined a cross linking of about 50 proteins (inset in Figure 3). This scattering was interpreted in terms of a Gaussian linear chain with relatively large statistical segments. In summary, we give a first clue concerning the role of protein mediated CaCO3 mineralization. Ovalbumin first acts as a cation sponge which 2+ locally increases the Ca concentration. These Carich pockets of ovalbumin aggregates seem to build nucleation centers for the incipient calcium carbonate formation. Calcium carbonate nanocrystals are stabilized by surface bound ovalbumin which is eventually occluded between the individual CaCO3 crystallites, thereby forming a mesocrystal, i.e. an inorganic-organic hybrid material [4]. The observed formation from less dense to more dense polymorphs follows the Ostwald-Volmer rule [1].

TAB.: SANS parameters of the calcium carbonate polymorphs, ovalbumin, and water.

compact structure of the native state. The slight increase of scattering after 90 minutes is the result of a 14.6% increase in the protein scattering length density which can be interpreted as a loading of the protein with about 280 Ca2+ ions. After 90 minutes a gradual transition to a Q-1 power law is observed which was complete after nearly 3 h (see also Figure 1); the structural change of the protein was a continuous process during this time interval. The strong increase in intensity at low Q indicated that the process was accompanied by a crosslinking ("salting out") of the protein molecules. In this time interval the formation of the amorphous polymorph proceeded and approached its maximum (Figure 2). After the protein was salted out, the amorphous phase started to dissolve as

[1]

S. Mann, Biomineralization, Oxford University Press, Oxford 2001. A. Heiss et al., Biointerphases 2 (2007) 16-20. V. Pipich et al., J. Am. Chem. Soc. (JACS) 130 (2008) 6879-6892. D. Schwahn et al., J. Phys. Chem. C111 (2007) 3224-3227.

[2] [3]

[4]

35

Kinetics of Micelle Formation

L. Willner1, R. Lund2, M. Monkenbusch1, P. Panine3, T. Narayanan3, J. Colmenero2 , D. Richter1
1 2

IFF-5: Neutron Scattering Donostia International Physics Center (DIPC), Donostia - San Sebastian, Spain 3 European Synchrotron Radiation Facility, Grenoble,France. The route by which amphiphilic molecules selfassemble into nano-scale objects such as micelles is still not fully understood. The formation of block copolymer micelles in selective solvents occurs spontaneously usually in the subsecond range. By means of synchrotron x-ray scattering with millisecond time resolution we got direct structural information in-situ on the birth and growth of block copolymer micelles. Using a quantitative model we showed that the self-assembly process can be viewed as a primary micellization and growth process where the elementary growth mechanism is the exchange of single unimers A classical example and a model system for selfassembly are amphiphilic diblock copolymers that undergo micellization in aqueous solution [1]. The morphology of such systems has been widely studied during the past, a detailed understanding of the mechanism and kinetic pathways of the selfassembly process has not been reached to date. This is primarily due to the lack of experimental techniques having the correct spatial and temporal resolution with the combination of a suitable welldefined model system. In addition there is a prominent lack of detailed physical modelling of the data. Thus so far, results remain largely inconclusive. In this work [2] we show that the required nanoscale spatial resolution and millisecond temporal resolution could be achieved for an in situ investigation by using synchrotron x-ray scattering. The self assembly process of a model amphiphilic block copolymer system was triggered by an interfacial tension jump experiment by rapidly changing the solvent quality for one of the blocks. Using a detailed quantitative model we further demonstrate that the kinetic pathway proceeds by unimer exchange where only single chains are added or removed at a time. As a model system we employed a well-defined poly(ethylene-alt-propylene -poly(ethylene oxide) (PEP1-PEO20, numbers indicate the approximate molecular weight in Kg/mole)block copolymer. This block copolymer forms starlike micelles in water and water/dimethylformamide(DMF)mixtures which are both selective solvents for PEO [3]. A large difference in interfacial tension, , with respect to PEPallows an effective tuning of the micellization properties by varying the solvent composition. In pure DMF only single chains (unimers) are present but as soon as some water is added the block copolymers spontaneously aggregate into micelles. This phenomenon was exploited by rapidly mixing a DMF solution with unimers with water/DMF pure solvent mixture by means of a stopped flow apparatus. The stopped flow set-up was coupled to the small angle x-ray scattering (SAXS) instrument at the high brilliance beamline, ID02, at the European Synchrotron Radiation Facility (ESRF) allowing a synchronization of extremely fast mixing (4.5ms) with rapid data acquisition. We chose an optimized acquisition time of 20 ms. A typical example of the time evolution of the scattering curves is presented in Figure 1. The data were obtained from a solution containing a total volume fraction of 0.25% block copolymer in a solvent mixture with 90 mole% DMF.
100 90 80 70 60 50 40 30 20 10 0

d/d/ (Q) [cm ]

14.5 ms 24.5 ms 194.5 ms 994.5 ms 2914.5 ms 240 s Unimer Reservoir Model fits

-1

0,01

Q [ ]
Fig. 1: Normalized absolute scattering cross sections at different times during the kinetics for the PEP-PEO system at a final polymer concentration of 0.25%. The solid lines display fit results from a standard core/shell model.

-1

0,02

0,03

The induced self-assembly process causes a strong increase in intensity directly reflecting the growth of the micelles in real time. The scattering data were analysed using a standard core shell model describing the detailed structural features of both the inner PEP core and the outer PEO corona of starlike micelles. The solid lines in Figure 1 represent fit results of the core/shell model. The excellent agreement indicates that at all times a starlike structure is adopted. An important parameter which can be extracted by the model fit is the mean aggregation number, Pmean. The growth of the micelles in terms of an increasing Pmean is shown in Figure 2 on logarithmic time scale for three different concentrations.

36

macroscopically determined quantities, e.g. the interfacial tension, , between PEP and water/DMF was found to be 19mNm compared to 12mNm obtained by pendant drop tensiometry. The kinetic pathway from unimers to the final micelle is schematically depicted in Figure 3. The initial free unimers are rapidly consumed in a primary micellization event as shown in region leading to classical overnucleation. Consequently, metastable micelles with a broader size distribution are obtained in region II corresponding to the shoulder in Pmean at intermediate times in Figure 2. A further growth of the micelles requires that some of them, particularly the smaller ones, disassemble to provide unimers. At later times in region III the equilibrium is approached with a narrow size distribution of the final micellar entity.

Fig. 2: Time dependence of the aggregation number, Pmean, extracted from the fits for three polymer volume fractionson a logarithmical time scale: 0.125% (stars), 0.25% (squares), and 0.5% (triangles). Solid lines represent a fit using the kinetical model described in the text.

II fast slow

III

Qualitatively the evolution of Pmean can be summarized as follows: At shortest times the data suggest the existence of a fast initial aggregation (t<5ms) that cannot be entirely resolved experimentally. This process becomes exhausted at intermediate times leading to a shoulder of Pmean that changes with concentration. The terminal relaxation towards a common equilibrium slows down with time. The overall rate increases with concentration. In order to quantitatively discuss the experimental data we have derived a kinetic model that involves simple unimer exchange as the single elementary growth step:

Figure 3: Schematic view of the kinetic pathway in the formation of polymeric micelles.

M P +U

k+

M P +1
k-

with MP as the number of micelles of size P and U the number of unimers. k+/k- denote the insertion and expulsion rate constants which both depend on P. Within the context of classical nucleation and growth theories [4,5] the formation and growth of the polymeric micelles is governed by the micellization potential, G(P,1) (1=unimer volume fraction),which in our case was taken as the difference of the free energy of a starlike micelle and an equivalent amount of unimers taking properly into account the translational entropy. Following Neu et al. [5] we assume the validity of the detailed balance or microscopic reversibility principle, which gives a net creation rate in terms of the concentration, P+1, and flux, jP+1:

In summary, the kinetics of formation of block copolymer micelles have been directly observed in situ by synchrotron small angle X-ray scattering with millisecond time resolution. Applying a quantitative model, we see that the formation and growth of micelles can accurately be described by a primary micellization and growth process governed by single unimer exchange mechanism. The contribution of other more complicated mechanisms, like fusion and fission, cannot entirely be excluded, however, the experimental data are sufficiently explained by insertion/expulsion of a single chain at a time.

[1] Hamley , I. W. The Physics of Block Copolymers, Oxford University Press, Oxford , UK (1998). [2] Lund, R. et al. submitted to Phys. Rev. Lett.. [3] Lund, R. et al. Macromolecules, 39,4566 (2006). [4] Nucleation Theory and Applications (J.W.P. Schmelzer ed.), Wiley-VCH Verlag GmbH & Co., KGaA, Weinheim (2005). [5] Neu, J.C. et al. Phys. Rev. E, 66, 061406 (2002).

jP +1 = k+ ( P)1[P P+1 exp(G(P + 1, 1 ) G(P, 1 ))]

which is only determined by the insertion rate constant k+ and the potential G(P,1). The whole micellar evolution can then be calculated by solving a system of differential equations which was done numerically by using standard routines and by fitting to the experimental data. Fit results are shown as solid lines in Figure 2. All concentrations could almost perfectly be described by a consistent set of parameters. The parameters nicely compare with

37

Polymer Chain Dynamics in a Random Environment

K. Niedzwiedz1, A. Wischnewski1 , M. Monkenbusch1, D. Richter1 , A.-C. Genix2 , A. Arbe3 , J. Colmenero2,3 , M. Strauch4 , E. Straube4
1 2

FZJ, Institute of Solid State Research - Neutron Scattering, Julich, Germany Donostia International Physics Center, Paseo Manuel de Lardizabal 4, 20018 San Sebastian, Spain 3 Centro de Fsica de Materiales CSIC-UPV/EHU, Apartado 1072, 20080 San Sebastian, Spain 4 Martin-Luther-Universitat Halle-Wittenberg, Fachbereich Physik, D-06099 Halle, Germany

The question of what determines the behaviour of two polymers which are perfectly miscible on the one hand, but very different in their dynamical properties on the other hand, is at present matter of controversial discussion. We present a neutron scattering study on the miscible polymer blend poly(ethylene oxide)/ poly(methyl methacrylate) (PEO/PMMA) which due to the very different time scales of motion is a perfect candidate to address this topic. Only exploring different time and length scales allowed to identify the mechanisms of dynamic heterogeneity on a molecular scale.

ment (msd) of the PEO segments, the experiments were performed at the instrument BSS at the FZJ in Julich.

The dynamics of a polymer chain in the melt is well described by the Rouse model [1]. Here, the relaxation of thermally activated uctuations is balanced by entropic and viscous forces (friction). The relevant parameter in this model is the friction of the observed chain in a heat bath representing the adjacent chains. One important consequence of the fact, that the segments of a chain are linked to each other (chain-connectivity), is that segments exhibit a mean squared displacement proportional to the square root of time different to the displacement for a Fickian diffusion which is increasing linearly in time. At present the investigation of dynamic miscibility in a blend of two different polymers is an active area of research. One of the important questions is how the segmental friction arises if a segment is surrounded by a chemically heterogeneous environment. The system PEO / PMMA is in particular interesting because of the signicantly different glass transition P P temperatures (Tg EO 200 K, Tg M M A 400 K). In such a situation, for temperatures around and beP low Tg M M A , PEO moves in the random environment created by the frozen PMMA component [2]. Starry-eyed, one could assume that in a perfectly miscible polymer blend the friction of a given segment is determined by the average friction of the blend components. We have investigated the PEO dynamics in PMMA by quasielastic neutron scattering. This has been realized by preparing a mixture of protonated PEO chains in deuterated PMMA. Samples with different concentrations (25%, 35% and 50% of PEO) have been measured at different temperatures between 350 K and 400 K. By neutron backscattering spectroscopy we have followed the mean square displace-

FIG. 1:

Mean square displacements obtained from backscattering spectra from the 35% PEO/PMMA sample at different Q and temperatures.

Fig. 1 shows the result for a sample with 35% protonated PEO in 65% deuterated PMMA at two different momentum transfers Q. The data follow nicely straight lines when plotted against the square root of time in good agreement with the Rouse prediction. Depending on temperature, the maximum displacement of a PEO segment observed during about 1 ns amounts to less than 1 nm. The related values for the friction can be obtained from the slope of these lines. They show a temperature dependence very similar to the one found in pure PEO, however the friction is about one order of magnitude larger than in the pure system. So far we may state that the dynamics of PEO in a matrix of PMMA at temperatures where PMMA is nearly frozen is in good agreement with Rouse dynamics characterized by an average friciton. From this one could conclude that the effect of blending is not more than imposing a different friction to a given segment, and therefore that the above mentioned naive assumption is true. Fig. 2 displays neutron-spin-echo (NSE) measurements of the single chain dynamic structure factor of PEO chains in the PMMA matrix measured at the IN15 (ILL, Grenoble) up to about 80 ns. In order to achieve this result a deuterated PMMA matrix was used, where a mixture of deuterated and protonated PEO was immersed. The dotted lines in Fig. 2 are

38

a result of a Rouse description based on the average Rouse friction obtained from the backscattering data in g. 1. Obviously this description fails grossly predicting a by far too fast decay. Apparently the motion is strongly slowed down towards longer times or larger length scales. We may roughly quantify this slowing down in tting an effective smaller Rouse rate. This reveals retardations by factors 4 to 20 (depending on PEO-content and temperature) compared to the 1 ns scale and gives clear evidence for an additional effect of blending which has not been seen in the BSS data. Finally, g.3 shows Fourier transformed time-of-ight (TOF) data on the same blend measured at the FOCUS instrument (PSI, Villigen) at T=400 K. For the blend also the backscattering data are included. At high Q, quasielastic neutron scattering observes local processes. Compared to the spectrum from pure PEO, the blend data are strongly broadened demonstrating a broad distribution of local relaxation processes in the PMMA environment. From the decay we realize that a shift of the characteristic time from 1 ps in pure PEO to about 6 ps for PEO/PMMA is found in excellent agreement with NMR data [3] and also with the ratio of the corresponding Rouse rates as determined by the backscattering experiment. However, the question remains where the extremely broad distribution comes from.

the backscattering data, the only adjustable parameter being its width. The solid lines in Fig. 2 show the best result which could be achieved for the 35 % PEO/PMMA sample. The three lowest Q values are in very good agreement with the experimental data, however, the highest Q data are reproduced at very short times only. The origin of this signicant deviation at the highest Q and longer times is still unclear, though it might have to be related to additional connement effects imposed by the matrix. The values of the width obtained by applying the same procedure to the other compositions and temperatures show the expected behaviour: the width of the distribution function increases with increasing PMMA concentration (in the limit of zero PMMA concentration it should go to zero) and within one blend it increases with decreasing temperature. Having in mind that at high Q we observe directly the distribution of relaxation times, we now can try to describe the TOF spectra of the blend displayed in Fig. 3. Starting from the pure PEO spectrum we shift the relaxation rate by a factor obtained from the backscattering data for the blend. In a second step we convolve the thus shifted PEO spectra with the log-normal distribution function using the result from the NSE data. With this we obtain the solid line in Fig. 3 in perfect agreement with the observed spectrum. Having in mind that all parameters have been obtained from other measurements, the width from the NSE-data and the shift of the relaxation rate from the BSS data, this agreement which combines data over 5 orders of magnitude in time is remarkable.

FIG. 2:

Single chain dynamic structure factor of the 35% PEO/PMMA system at T=400 K. Q-values are 1 nm1 , 1.5 nm1 , 2 nm1 and 3 nm1 from top to bottom. The dashed base represents the elastic contribution to the scattering. The dotted lines illustrate the Rouse theory with Rouse relaxation rate obtained from the backscattering data. The solid lines show the results of the model with randomly distributed friction.

FIG. 3: Fourier transformed TOF data of pure PEO and

35% PEO/PMMA at T = 400 K. For the blend system also backscattering data are included. The solid line through the PEO data describes a t with a stretched exponential ( = 0.5). Line through the blend data: see text.

In the following we demonstrate that the contradictory experimental ndings can be explained assuming that the mobile PEO chain experiences a heterogeneous environment imposed by the rather stiff PMMA matrix. This picture can be rationalized by a chain where the beads undergo a random friction with a distribution of friction coefcients. The dynamic structure factor for such a chain has been calculated by starting with the Langevin equation for a polymer chain and introducing a set of bead mobilities following a log-normal distribution centered around the average mobility obtained from

[1] P. E. Rouse, J. Chem. Phys. 21, 1272 (1953) [2] A.-C. Genix, A. Arbe,F. Alvarez, J. Colmenero, L. Willner and D. Richter, Phys. Rev. E 72, 031808 (2005) [3] T. R. Lutz, Y. He, M. D. Ediger, H. Cao, G. Lin and A. A. Jones, Macromolecules 36, 1724 (2003)

39

JCNS Instrumentation
T. Gutberlet 1
1

JCNS: Jlich Centre for Neutron Science

On May 2nd , 2006 the FRJ-2 research reactor at Forschungszentrum Jlich GmbH was shut down ending a successful scientic work of more than four decades. The end of this research facility with neutrons marked at the same time the start of a new era for research with neutrons at Forschungszentrum Jlich GmbH with the timely establishment of the Jlich Centre for Neutron Sciences (JCNS) on February 16th , 2006. At the national and international leading sources FRM II, ILL and SNS JCNS operates state-of-the art neutron instruments under a common scientic objective. Instruments operated by JCNS are open to scientists from Germany, Europe and across the globe for the benet of their scientic research goal. Access is organized by the JCNS User Ofce.

FIG. 1: View of neutron guide hall of FRM II. In addition to the relocated instruments the second generation backscattering spectrometer with phase space transformer SPHERES was built at FRM II in the frame of the "Verbundforschung" funding of the BMBF. Regular operation and access or external users started in March 2008. At current SPHERES offers an energy resolution of 0.65 eV with a signal to noise ratio of better as 600:1. JCNS is further constructing four more brand-new instruments at FRM II: together with the Technical University of Munich, a diffractometer for biological structure determination BIODIFF is being built till 2010. A brand-new high intensity magnetism reectometer MARIA is in construction to be operational in 2009. The new thermal time-of-ight spectrometer TOPAS, replacing the former SV-29 of FRJ-2, and the new time-of-ight powder and texture diffractometer POWTEX will be installed at the new guide hall east of FRM II. Both instruments are expected to be operational in 2011. POWTEX is realised and nanced in the frame of the BMBF "Verbundforschung" and build in a collaboration of RWTH Aachen University and the Forschungszentrum Jlich within the Jlich Aachen Research Alliance JARA with the Gttingen University as further partner. This instrument is designed to serve the need of the solid state chemistry and geoscience communities. In addition of developing, constructing and running high class neutron instruments at FRM II JCNS also offers users specialized and unique sample environment and laboratory facilities on-side. Modern chemistry and biology labs can be used for sample characterization and sample preparation. In addition to the FRM II location, JCNS operates outstations at the rst MW spallation source SNS in Oak

The JCNS reects the unique role of Forschungszentrum Jlich GmbH within the German neutron scattering landscape based on a compelling in-house condensed matter research program focussing on neutron scattering in the areas soft condensed and biological matter and nanomagnetism and highly correlated electron systems. Some of the best instruments from the closed DIDO reactor were transferred to the new national neutron source FRM II in Garching close to Munich (Fig. 1). Besides the necessary adaptations of the instruments, a rigorous upgrade and renewal programme was embarked, which comprised of up to 13 instruments. A new branch lab with 32 scientists and technicians has been established at the new research reactor FRM II in Garching since 2006. Transferred instruments included the powerful small angle neutron scattering units KWS-1 and KWS-2, the novel focussing VSANS machine KWS-3, the Jlich neutron spin echo spectrometer J-NSE, the diffuse polarized neutron spectrometer DNS and the reectometer HADAS. HADAS was transferred to FRM II to serve as test and development instrument TREFF. DNS, KWS-2, and J-NSE have been in operation for users since autumn 2007 now. The second SANS KWS-1 and the focusing SANS KWS-3 will become available for users soon. KWS-3 has been rebuild and up-dated including a brand new mirror coating and will be available for users till end of 2009.

40

Instrument BIODIF

Facility FRM II

DNS J-NSE

FRM II FRM II

KWS-1

FRM II

FIG. 2: Inside of the spin echo spectrometer at SNS. Ridge, USA and the highest ux neutron research reactor of the Institute Laue-Langevin ILL in Grenoble, France. At SNS, the erection of the ultra high resolution neutron spin echo spectrometer is nearing completion (Fig. 2). This next generation spin echo spectrometer will offer unprecedented resolution and dynamical range. This investment in the instrumentation of the SNS gives German users access to two further high class instruments: the backscattering spectrometer BASIS and the high resolution powder diffractometer POWGEN3. Within a CRG contract with ILL concerning the cold triple axis spectrometer IN12 is operated in a consortium together with the CEA Grenoble. This engagement also provides German users additional access to the thermal triple axis spectrometer IN22 and the lifting counter diffractometer D23. In the framework of the ILL Millennium programme, IN12 will be relocated to a new guide end position and JCNS has committed itself to a complete overhaul of the instrument, which will lead to signicant improved performance. An overview of JCNS operated instruments is given in Table 1. JCNS instruments are open to scientists from Germany, Europe and across the globe for the benet of their scientic research goals. JCNS gratefully acknowledges complementary funding support from the EU within the 6th Framework Programme through the Key Action: Strengthening the European Research Area, Research Infrastructures in the project NMI3 - Integrated Infrastructure Initiative for Neutron Scattering and Muon Spectroscopy. 2/3 of the available beam time at JCNS instruments is distributed to external users by application for beam time reviewed by an international Scientic Review Committee twice a year. Figure 3 shows the distribution of external proposals received in the four proposal rounds in 2007 and 2008 for the instruments of JCNS. Concerning the number of proposals the small-angle scattering machine KWS-2 clearly stands out. With the various instruments at FRM II, ILL and SNS JCNS provides external users unique access to world class instruments under standardized conditions at the neutron source best suited for their respective ex-

KWS-2 KWS-3 MARIA POWTEX SPHERES

FRM II FRM II FRM II FRM II FRM II

TOPAS

FRM II

TREFF IN12 SNS-NSE

FRM II ILL SNS

Parameters cold neutrons protein crystallography cold neutrons polarisation analysis Fourier times 2 ps (4.5 ) < < 350 ns (16 ) high resolution GISANS polarisation analysis high intensity 104 < q < 103 1 high intensity polarisation analysis middle resolution texture diractometer energy resolution 0.65 eV q = 0.16 to 1.84 1 energy transfer up to 100 meV polarisation analysis polarisation analysis polarisation analysis Fourier time up to 1 sec

TAB. 1: JCNS instruments.

FIG. 3: Number of proposals submitted in the year 2007

and 2008 on the neutron scattering instruments of JCNS.

periments. Further information on the instruments of JCNS and Call for proposals for beam time can be found on the JCNS web pages at www.jcns.info.

41

42

IFF-7: Soft Condensed Matter

Director: Prof. Jan K. G. Dhont

The Soft Condensed Matter group investigates the chemistry and physics of colloidal systems. Colloidal systems can be regarded as solutions of very large molecules which exhibit phase transitions and show non-equilibrium phenomena that are also found for simple molecular systems. Due to the slow dynamics of colloids and the tuneable interactions between the colloidal particles, however, there are many transitions and non-equilibrium phenomena that do not occur in simple molecular systems, like gellation and shearband formation. The aim is to understand structure, dynamics and non-equilibrium phenomena on a microscopic basis with an open eye for possible technological applications.

The main topics that are studied include, the phase behaviour, pattern formation, phase separation kinetics and dynamics of suspensions of spherical and rod-like colloids under shear flow, mass transport gradients, induced by temperature

dynamics and micro-structural properties of colloidal systems near walls and interfaces, the effects of pressure on interactions, the location of phase transition lines and gellation transitions and the dynamics of colloids and polymers, response of colloids to external electric fields, the equilibrium phase behaviour of mixtures of colloids and polymer-like systems, dynamics of various types of colloidal systems in equilibrium, and the synthesis of new colloidal model particles, with specific surface properties, interaction potentials and particle geometries.

43

Tracer-Sphere Diffusion in Rod-Networks

K. Kang 1 , A. Patkowski 2 , J. K. G. Dhont 1
1 2

IFF-7: Soft Condensed Matter A. Mickiewics University, Poznan, Poland

Mass transport of colloidal-like particles through networks is relevant for a number of separation-, purication- and characterization techniques of macromolecular mixtures and might play a role in diffusive transport (of proteins) through crowded environments in cells. To gain in understanding on the physics that underlies the transport characteristics through such conning media, translational diffusion of tracer spheres in isotropic and nematic networks formed by long and thin colloidal rods is investigated as a function of ionic strength and rod concentration. In particular, the hydrodynamic screening length of isotropic and nematic rod-networks is determined from a newly developed theory and experimental tracer-diffusion data obtained with Fluorescence Correlation Spectroscopy (FCS). The majority of reported tracer-diffusion experiments of spherical particles in rod networks focus on proteins in suspensions of F-actin, which are relevant for mass transport in cells. Recently, experiments on tracer diffusion of spheres in host suspensions of slender particles, other than F-actin, have been reported : nucleosome core particles in dispersions of DNA [1], colloidal spheres in solutions of "living polymers" [2] and colloidal spheres in dispersions of xanthan [3]. The host networks in these references exhibit a quite complicated dynamics by themselves. As far as we know, there are no experimental data available on tracer diffusion of colloidal spheres in the much more simple quasi-static networks consisting of relatively stiff, very long and thin rods, aside from three earlier papers of the present authors [4][6]. Besides experimental work, an attempt has been made in these papers to develop a microscopic theory for tracer diffusion of spheres through networks of stiff, long and thin rods, where both (screened) hydrodynamic interactions and direct interactions between the tracer sphere and the rod network are explicitly accounted for.

FIG. 1: The two extreme cases where (a) the tracer sphere is large compared to the mesh size of the network, and (b) where the sphere is small compared to the mesh size.

EHUT). Moreover, due to the very small van der Waals attractions between the fd-virus particles, the rod networks are stable over a wide range of salt concentrations, which offers the possibility to study diffusion in these networks as a function of the range of electrostatic interactions. There are fundamentally different mechanisms leading to slowing down of diffusion of a tracer sphere due to the presence of a rod network, depending on whether the tracer sphere is large or small in comparison to the mesh size of the network. For large tracer spheres, translational motion of the tracer sphere is only possible when the network structure is severely distorted (see Fig.1a). In this case, direct interactions between the sphere and the rods is much more pronounced than hydrodynamic interactions. Tracer diffusion can then be described with the neglect of hydrodynamic interactions [4]. For small tracer spheres, where the sphere can move through the voids of the network without distorting the structure of the network (see Fig.1b), hydrodynamic interactions are important. Due to entanglement of the rods in the network, hydrodynamic interactions between the tracer sphere and the rods are screened. This screening is quantied by the so-called hydrodynamic screening length 1 , which measures the penetration depth of shear waves into the network. For the rod networks considered here, there is no theory available yet for the hydrodynamic screening length. Despite

We use fd virus as a rod-like colloidal host particle, which is indeed a very long and thin, rather stiff rod (length 880 nm, thickness 7 nm and persistence length 2500 nm), and does not exhibit polymerization/de-polymerization nor possible bundle formation (like F-actin, wormlike micelles and

44

the fact that the network structure remains essentially unchanged during the diffusive motion of such a small sphere, there is nevertheless a distortion of the pair-correlation function between the sphere and the rods. This distortion is due to the fact that, when the sphere moves past a rod, the probability to nd the sphere on the side-of-approach of the rod is larger than that on the "shadow side" of the rod. The rod-sphere pair-correlation function is therefore distorted, giving rise to a force on the sphere that affects its diffusive motion. We referred to the distortion of the pair-correlation function in ref.[5] as "the shadowing effect". The shadowing contribution is not only determined by hard-core interactions, but also by charge-charge interactions in case the rods and the sphere carry surface charges. The importance of the shadowing effect relative to hydrodynamic interactions can thus be varied systematically by changing the electrostatic screening length through variation of the ionic strength of the rod suspensions [6].

FIG. 3: (a) The hydrodynamic screening length 1 in units of the rod length L for the isotropic state versus the fdconcentration. The symbols refer to the different buffer concentrations : cT = 0.16 mM , 0.80 mM , 4.00 mM , 20.0 mM , 20 mM + 100 mM N aCl . (b) The screening length for the highest buffer concentration and 100 mM added salt in the isotropic and the nematic phase versus the fd-concentration.

FIG. 2: Long-time self diffusion coefcients of apoferritin in fd-virus particle suspensions for two ionic strengths. (a) TRIS-buffer concentration of 0.16 mM (from ref.[6]) and (b) 20 mM plus 100 mM added N aCl (from ref.[5]). The vertical grey bars indicate the two-phase, isotropic-nematic coexistence region.

in Fig.3a. The screening length 1 for various ionic strengths scale onto a single master curve, and decreases with increasing fd-concentration, as expected. The hydrodynamic screening lengths for the nematic phase are given in Fig.3b. Here, the distinction between directions perpendicular and parallel to the nematic director should be made. Surprisingly, the hydrodynamic screening length increases with increasing fd-concentration in the nematic state. This is probably due to the increasing degree of alignment of the rods with increasing concentration. It thus seems that the hydrodynamic properties of nematic networks is highly sensitive on the orientational degree of order. This is conrmed from measurements of 1 at a xed fd-concentration but different orientational order parameter through the variation of ionic strength [6].

Tracer diffusion constants of apoferritin in fd-virus suspensions were measured with Fluorescence Correlation Spectroscopy (FCS). Since fd-virus particles uoresce to some extent, the resulting contribution to the correlation function must be subtracted [5]. The creation of mono-domain nematics through alignment in a magnetic eld and the measurement of orientational order parameters of the nematic samples is discussed in that reference. In Fig.2 the measured diffusion coefcients are plotted as a function of fd-concentration for two analytical TRIS/HCl-buffer concentrations that are used (0.16 mM , and 20.0 mM plus 100 mM N aCl). As can be seen, there is a strong effect of the ionic strength on the diffusive behaviour of apoferritin. The way to extract hydrodynamic screening lengths from the diffusion data, based on the newly developed theory, is extensively discussed in ref.[6]. The hydrodynamic screening length as a function of the fd-concentration for isotropic networks is shown

[1] S. Mangenot, S. Keller, J. Rdler, Biophys. J. 85, 1817 (2003). [2] J. van der Gucht, N.A.M. Besseling, W. Knoben, L. Bouteiller, M.A. Cohen Stuart, Phys. Rev. E 67, 051106 (2003). [3] G.H. Koenderink, S. Sacanna, D.G.A. Aarts, A.P. Philipse, Phys. Rev. E 69 (2004) 021804. [4] K. Kang, J. Gapinski, M.P. Lettinga, J. Buitenhuis, G. Meier, M. Ratajczyk, J.K.G. Dhont, A. Patkowski, J. Chem. Phys. 122, 044905 (2005). [5] K. Kang, A. Wilk, J. Buitenhuis, A. Patkowski, J.K.G. Dhont, J. Chem. Phys. 124, 044907 (2006). [6] K. Kang, A. Wilk, A. Patkowski, J.K.G. Dhont, J. Chem. Phys. 126, 214501 (2007).

45

Enhanced Slowing Down of the Colloidal Near Wall Dynamics in a Suspension of Rods
P. Holmqvist, D. Kleshchanok, P. R. Lang
IFF-7: Soft Condensed Matter

In this report, we will show the inuence of rodlike particles, fd-virus, on the diffusion of spherical polystyrene colloids close to a wall. The sphere diffusivity normal to the wall, < D >, is strongly affected by the presence of the rods, while the effect on the parallel diffusivity, < D >, is less pronounced except in the immediate vicinity of the wall [1]. The slowing down and the anisotropy of Brownian motion close to a wall due to hydrodynamic drag forces has been theoretically predicted [2] and recently experimentally veried [3]. So far, only the case of particles interacting by excluded volume with a wall has been considered. The effect of depletion on the dynamics close to a wall has received very little or no attention, neither theoretical nor experimental.

independently [3]. The depletion potential mediated by rodlike particles has been calculated by Mao et al. to third order in rod number density [5]. In the rst order approximation, there is a simple closed analytical form, which can be introduced in the expressions for the initial relaxation rate of the time auto correlation function of the scattered intensity. Our experimental TIRM data for the depletion potential mediated by fdvirus is in quantitative agreement with these predictions. Therefore, we used the rst order approximation to estimate the lowest rod concentration at which the depletion potential would cause a notable effect on the near wall dynamics of spherical colloids with a radius of R = 85 nm. We note that the Derjaguin approximation is not valid at this radius/rod length ratio, but it will give a lower boundary for the required rod number density, because the strength of the depletion potential decreases with decreasing R/L at constant density. From this, we chose as a starting point a rod concentration of twice the overlap concentration which would give a contact potential of approximately 0.5kB T . Further, at this rod concentration, the solvent viscosity will be changed only by a few percent and the fdvirus contribution to the scattering is essentially negligible, which keeps the data treatment on a tractable level. To our surprise, we found a much larger effect on the near wall particle dynamics than expected from these considerations. Aqueous buffer solutions (20 mM TRIS) of PS latex spheres with a radius of R = 85 nm were investigated with two different fdvirus concentrations, that is, 0.05 and 0.17 g/L, above and below the overlap concentration, c = 0.075 g/L. In bulk solutions with the same fdvirus content, the diffusion of the colloids is reduced by less than 20% [6]. The PS latex spheres are charge stabilized by sulfonate surface groups, and they were diluted from their stock solutions to a volume fraction of 2 104 . At these conditions, the particles may be regarded as hard spheres, since the Debye screening length is in the range of 3 nm, while the mean interparticle distance is of the order of several thousand nanometers. A schematic picture of the system close to the surface under evanescent illumination can be seen in gure 1. To illustrate the effect of the fd-viruses on the colloidal diffusion close to the wall correlation functions g1 (t) are presented in gure 2 at a constant penetration depth of 2/ = 270 nm and a total scattering

FIG. 1: Schematic picture of the sphere/rod system under evanescent illumination close to the surface

We chose to apply fdvirus as a depletant, which are rodlike mono-disperse particles and nonadsorbing neither on glass nor on polystyrene latex particles. They have a contour length of 880 nm which is comparable to the maximum penetration depth applicable in an EWDLS experiment. In other words, the depletion potential mediated by fdvirus is expected to be effective throughout the entire scattering volume. Further, we performed EWDLS measurements with our tipple axis setup, with which we can determine the parallel and normal component of the diffusivity

46

FIG. 2: Initial decay ln(g1 (t)) for 2 104 volume fraction PS latex spheres (R = 85nm) in cf d = 0.17 g/L at Qtot = 0.0157 nm1 and 2/ = 270 nm for bulk (open squares) and for two different combinations of Q and Q as follows: Q = 0.0136 nm1 and Q = 0.00785 nm1 (open circles ), and Q = 0.00785 nm1 and Q = 0.0136 nm1 (open triangles). The lines are the expected ln(g1 (t)) for the same system without fdviruses for bulk (black), for Q = 0.0136 nm1 and Q = 0.00785 nm1 (red), and for Q = 0.00785 nm1 and Q = 0.0136 nm1 (green).

FIG. 3: < D > (squares) and < D > (circles) normalized to the bulk diffusion, D0 , are plotted against the reduced penetration depth, 2/R, for cf d = 0.05 g/L (solid symbols) and cf d = 0.17 g/L (open symbols). The lines are the expected curves for the colloid solution without depletant.

vector, Qtot = 0.0157 nm1 , for two different combinations of Q and Q together with the bulk g1 (t) for a fdvirus concentration of 0.17 g/L. The EWDLS g1 (t)) show a much smaller slope than that of the bulk curve. Further, the slope of the g1 (t) at Q > Q is signicantly smaller than that at Q < Q , which indicates a strong anisotropy of the near wall diffusion. If these data are compared to the expected g1 (t) for the same system without depletant [3] (lines in gure 2), it is clear that the sphere near wall dynamics is affected much more than the bulk dynamics by the presence of the rods. In bulk, the g1 (t) values with and without the fdvirus are almost identical (the 20% reduction is hardly measurable), while a large difference is obvious for the EWDLS correlation functions. Not only do the near wall dynamics slow down signicantly, but the anisotropy increases. This slowing down of the diffusion close to the surface cannot be explained solely by the small reduction of the bulk diffusion. To investigate this in detail, systematic measurements of the diffusivity parallel and normal to the surface at different penetration depths were performed. Using the same procedure described in our recent papers [3] the mean diffusivities for the parallel, < D >, and the normal diffusion, < D >, is determined for the two different fdvirus concentrations. To get a more complete picture of the effect of fdvirus on the colloidal dynamics close to the wall, the procedure described above was performed for a series of six penetration depths. In gure 3, the extracted mean diffusivities normalized to the bulk diffusion constant, D0 , are plotted against the reduced penetration depth, 2/R (where R is the colloidal radius). The anisotropy of the diffusion can nicely be seen as the separation between < D > (black squares) and < D > (red circles) for both concentrations, 0.05 g/L (solid sym-

bols) and 0.17 g/L (open symbols). For comparison, we show the expected curves for the colloid solution without fdvirus as solid lines. It is notable that the anisotropy is always larger for the high fdvirus concentration, while for the low fd-virus concentration the anisotropy is close to what can be expected for a system without depletant. If one compares the data with the expected mean diffusivities without depletant (solid lines), no effect on < D > can be seen at large penetration depths for both fdvirus concentrations. However, at penetration depths below about 450 nm, that is, 2/R 5, a pronounced effect can be seen for both fdvirus concentrations. < D > is decreasing much more with decreasing penetration depth than was observed for the system where no depletant was present. This effect is more pronounced in the solution with the high fdvirus concentration. If one looks at the normal diffusivity, < D >, a signicant reduction can be seen as compared to the nondepletant system also at large penetration depths (distances) for both fdvirus concentrations. As for < D >, < D > decreases with decreasing penetration depth, and for the high fdvirus concentration system < D , > drop more strongly at low penetration depths.

[1] Holmqvist P.; Kleshchanok, D.; Lang, P. R. Langmuir 23, 12010 (2007). [2] Brenner, H. Chem. Eng. Sci. 16, 242 (1961). [3] Holmqvist, P.; Dhont, J. K. G.; Lang, P. R. Phys. Rev. E 74, 021402 (2006). [4] Kihm, K. D.; Banerjee, A.; Choi, C. K.; Tagaki, T. Exp. Fluids 37, 811 (2004). [5] Mao, Y.; Cates, M. E.; Lekkerkerker, H. N. W. J. Chem. Phys. 106, 3721 (1997). [6] Kang, K.; Gapinski, J.; Lettinga, M. P.; Buitenhuis, J.; Meier, G.; Ratajczyk, M.; Dhont, J. K. G.; Patkowski, A. J. Chem. Phys. 122, 044905 (2005).

47

Dynamics in colloidal suspensions: from neutral to charged particles

G. Ngele 1 , A.J. Banchio 2
1 2

IFF-7: Soft Condensed Matter Universidad Nacional de Crdoba, Argentina

We have made a comprehensive study on the dynamics of suspensions of charge-stabilized and neutral colloidal spheres. Numerous short-time dynamic properties including diffusion functions, translational and rotational self-diffusion coefcients and the high-frequency viscosity have been computed by means of a powerful accelerated Stokesian Dynamics simulation method. The results of this study were used, in particular, to explore the validity of generalized StokesEinstein relations, and the possibility of measuring self-diffusion in a scattering experiment at an experimentally accessible wavenumber.

The dynamics of suspensions of charged colloidal particles is of fundamental interest in soft matter science, surface chemistry and food science. The scope for these systems has been broadened even further through the increasing importance of biophysical research dealing with charged biomolecules such as proteins and DNA [1]. Many of the theoretical and computer simulation methods developed in colloid physics are applicable as well to biological molecules and cells. In addition, suitably modied methods from colloidal physics can also be applied to dispersions of very small, i.e., nanosized particles not much bigger than the solvent molecules. Charged colloidal particles interact with each other directly by means of a screened electrosteric repulsion, and through solvent-mediated hydrodynamic interactions (HIs). These interactions cause challenging problems in the theory and computer simulation of the colloid dynamics. In this communication, we report on extensive Stokesian Dynamics (SD) simulations where the inuence of electrosteric and hydrodynamic interactions has been studied for numerous short-time properties including the high-frequency viscosity , the wavenumber-dependent diffusion function D(q) determined in a scattering experiment, the so-called hydrodynamic function H(q), and the rotational and translational short-time self-diffusion coefcients Dr and Ds , respectively. The simulations have been performed, with a full account of the many-body HIs, in the framework of the model of dressed spherical macroions interacting by an effective pair potential of screened Coulomb type [2, 3]. A large variety of sys-

Simulation test of short-time GSE relations relating the rotational and translational self-diffusion coefcients Ds and Dr , and the cage diffusion coefcient, D(qm ), respectively, to the high-frequency shear viscosity . All depicted quantities are normalized by their zero concentration values marked by the subindex 0. Symbols: SD simulation data. Lines: theoretical results. (a) Charged particles in salt-free solvent (water). (b) Suspension of neutral colloidal spheres. From [2].
FIG. 1:

tems with differing particle concentrations, charges and added salt concentrations have been considered, spanning the range from hard-sphere systems to low-salinity suspensions where the charged particles repel each other over long distances. Through comparison with the simulation data, the applicability and the level of accuracy of analytical methods

48

corresponding GSE relations. The general trends in the concentration and salt-dependence of the transport coefcients predicted in our SD simulations are in agreement with experimental ndings on chargestabilized suspensions and neutral particle systems. It has been suggested that the (short-time) selfdiffusion coefcient can be probed in a dynamic light scattering (DLS) experiment at a specic wavenumber qs located to the right of the principal peak in S(q), where S(qs ) = 1. The assumption made here is that the dynamic scattering function at such a wavenumber is essentially determined by selfdiffusion, without noticeable collective diffusion contributions. Indeed, if this assumption is valid at least on an approximate level, Ds D(qs ), where D(qs ) is the diffusion function measured at qs . Fig. 2 includes SD simulation results for S(q), D(q) and the hydrodynamic function H(q) for a salt-free suspension of charged particles. All dynamic quantities are normalized by the self-diffusion coefcient Ds . As can be noticed, Ds D(qs ) is obeyed indeed within reasonable accuracy. For all systems examined in our SD study, we nd the difference between D(qs ) to be less than ten percent, both for charged and neutral particles. Thus, DLS at the specic point qs can be used to obtain a decent estimate for the value of Ds . This nding is of relevance in numerous scattering experiments on colloidal systems where the large wavenumber regime is not accessible experimentally, and where alternative techniques to measure Ds directly are not applicable or unavailable. Colloidal hard spheres have a common static and hydrodynamic length scale set by the particle radius a. This leads to the occurrence of an isobestic wavenumber qa 4.02 where both S(q) and D(q)/Ds are equal to one, independent of the volume fraction. In low-salinity suspensions, the geometric mean particle distance becomes another length scale of physical relevance. Consequently, in these systems there is no isobestic point for S(q) and H(q). The non-existence of a -independent isobestic point in salt-free suspensions is exemplied in Fig. 2. In summary, our comprehensive SD simulation study of short-time transport properties forms a useful database for researchers interested in information on the suspension dynamics of globular colloidal particles, from systems of large, micron-sized colloids down to proteins in the nanometer range such as lyzozyme and apoferritin. A simulation analysis of the long-time dynamics of charged colloidal spheres with a full account of the HIs remains as a major challenge which we plan to address in future work.

SD results for the static structure factor, S(q), normalized short-time diffusion function, D(q), and hydrodynamic function H(q) = S(q) D(q)/D0 of a salt-free suspension of charged particles, as a function of the wavenumber q times the particle radius a. The dynamic quantities D(q) and H(q) are scaled by the respective short-time selfdiffusion coefcient Ds . The dashed vertical lines mark the wavenumber qs where S(qs ) = 1. From [3].
FIG. 2:

and ad-hoc concepts has been tested, which are frequently applied in the description of the colloid dynamics. As an important application, the SD simulation scheme has been used to scrutinize the validity of short-time generalized Stokes-Einstein (GSE) relations that provide an approximate link between diffusion coefcients and the high-frequency viscosity. GSE relations are fundamental to a growing number of microrheological experiments. The assessment of the quality of these relations is a necessary prerequisite for the experimentalist interested in deducing rheological information from dynamic scattering experiments. Our simulation results show that the accuracy of a GSE relation is strongly dependent on the range of the interaction potential, and the particle concentration. See Fig. 1 for an illustration of this important point where simulation results for the static structure factor S(q) at various volume fractions are compared to corresponding results for D(q) and H(q). The function H(q) is a direct measure of the HIs. Without HI, H(q) would be a constant equal to one, independent of the scattering wavenumber. A strictly valid GSE relation would be represented in Fig. 1 by a horizontal line of height equal to one, independent of the colloid volume fraction . As shown in this gure, the GSE relation for the cage diffusion coefcient, D(qm ), related to the position, qm , of the static structure factor peak, applies reasonably well to neutral hard spheres and to high-salinity systems (see Fig. 1b). However, it is strongly violated in the case of salt-free suspensions of charged particles (see Fig. 1a). Our simulations show further that rotational self-diffusion, and to a lesser extent also translational self-diffusion, are faster than predicted by the

[1] M.G. McPhie and G. Ngele, Phys. Rev. E 78, 78, 060401(R) (2008). [2] A.J. Banchio and G. Ngele, J. Chem. Phys. 128, 104903 (2008). [3] A.J. Banchio, M.G. McPhie and G. Ngele, J. Phys.: Condens. Matter 20, 404213 (2008).

49

Synthesis of silica rods, wires and bundles using lamentous fd virus as template
J. Buitenhuis, Z. Zhang
IFF-7: Soft Condensed Matter

We explored fd as a template to direct the formation of silica nanomaterials with different morphologies through simple sol-gel chemistry[1]. Depending on the conditions silica nanowires can be formed, which seem to accurately transcript the bending conformation and the length of the fd viruses in solution. But also surprisingly straight silica rods may be formed, and under other conditions bow-tie-shaped bundles of rods are formed, which have a remarkably well dened shape and dimension.

One dimensional anisotropic inorganic nanostructures such as tubes, rods, wires, bers, etc. are in the focus of research interests due to their potential applications, for example in optical, electronic and mechanical devices, sensors and catalysis[2, 3]. The synthesis of these anisotropic nanostructures is a big challenge, because most inorganic materials do not form the desired structure by themselves. In contrast to inorganic systems, biological and organic materials, especially supramolecular systems, usually have a well dened structure down to the nanoscale. Using (bio)organic materials as a template to build up anisotropic inorganic nanostructures has therefore emerged as a highly attractive method in recent years. The results described in the present paper may serve as a basis for the further development of the synthesis of inorganic materials using biopolymers as a template. In this paper, the lamentous fd virus is used as a template to regulate the formation of silica nanomaterials with well-dened morphologies. Fd viruses have a length of 880 nm and a diameter of 6.6 nm. M13, a virus which is almost identical to fd, differing only in one amino acid per coating protein, has been intensively explored by Belcher, Hammond and co-worker as a template in the synthesis of metallic and other magnetic and semiconducting nanowires[4]. Their strategy is to modify the coat protein of M13 via genetic engineering specically for each metal or oxide, so that the coat protein can selectively induce precipitation or assembly of that specic metal or oxide on the surface of the virus. However, as far as we know, there is no report concerning the application of fd or M13 as a template for silica precipitation. In contrast to the complicated genetic engineering

FIG. 1: TEM images of typical rods. (A) rods with a uniform

silica layer and semi-spherical ends, an assembly of three rods is indicated by the arrow; (B) a rod with a clear coreshell structure; (C) a slightly curved rod where no core-shell structure is visible; (D) EDAX analysis of the rod shown in C.

route used with the M13 virus, we show here that wild-type fd virus can also be used as a template in the synthesis of inorganic materials using simple sol-gel chemistry. Under different conditions, using acid-catalyzed hydrolyzation and condensation of tetraethoxysilane as silica precursor, three kinds of morphologies are observed: 1) silica nanorods with a diameter of 20 nm and a homogeneous silica layer, 2) nanowires with a curved shape and 3) bow-tieshaped bundles with well-dened shape and hierarchy. As far as we know, we are the rst to use fd as a template for material synthesis and have observed several interesting structures. Single silica rods with high uniformity, aggregated silica-virus hybrid nanorods as well as pronounced granular silica are observed for sample type 1, see g. 1. Along the axis of the rods, the diameter is constant and the silica layer is homogeneous. The surface of these rods is smooth under the maximum

50

FIG. 2: TEM of nanowires. A broken wire can be seen in

the inset as indicated by the arrow.

FIG. 3: TEM image of bow-tie-shaped bundles dispersed

in the background of granular silica. A possible subunit of the bundles, a silica fan is indicated by the arrow. Inset: schematic drawings of possible structures of the bundles.

resolution of the TEM we used and the shape of the ends of the rods is semi-spherical. Some of the rods show a clear core-shell structure with a low contrast part along the center of the whole rod (g. 1b). The low contrast part might be fd. However, some rods do not show such low contrast part and look like pure silica rods (g. 1c). The "pure" silica rod shown in g. 1c was subjected to EDAX analysis. Apart from silicon and oxygen, nitrogen and phosphorus are detected (g. 1d). The nitrogen and phosphorus can only be attributed to fd, given that no agents containing nitrogen or phosphorus were used during the synthesis. Although fd is semi-exible and somewhat curved in dispersion, most of the rods are straight and only a few slightly curved rods are seen (g. 1c). From a single rod point of view, the silica coating is highly uniform. Also the diameters of different rods are all close to the average value of about 20 nm. However, large differences in length are seen for different rods. Long rods with a length comparable to the length of intact fd are observed along with short rods, which might form from the silica coating of the fragments of decomposed fd. At somewhat different reaction conditions, long, curved wires are observed entangled with each other (sample type 2, g. 2). The surface of these wires is less smooth than that of the straight rods described before, and the diameter of these wires shows a less sharp distribution with an average diameter of about 23 nm. The contour length of these wires is in the range of that of intact fd, while long wires with a length twice that of fd are also observed. The longer wires seem to consist of two viruses sticking together by partial parallel overlap. These results imply that most fd remains intact during wire formation (an exception is the broken wire shown in the inset of g. 2 by the white arrow), in contrast to the case of the straight rods described above, where many rods much shorter than fd are observed. The curved shape of these wires probably originates from the bending congurations of the semi-exible fd virus in aqueous media. Therefore, these hybrid silica wires show an example of a quite precise transcription of the template, here, semi-exible fd. Whether or not the silica coating solidies the fd virus completely so

that the exibility is lost is not clear. Bow-tie-shaped bundles of silica rods are formed (g. 3) if the aqueous straight rod sample of type 1 is mixed with a methanol/ammonia mixture. This morphology is remarkable because as far as we know, no similar morphology has been reported for any other virus or organic template in the past. The bundles all have similar dimensions. The maximum length along the y axis of the well-dened bundles is about 2000 nm, comparable to the total length of two intact fd viruses joined with each other head-to-tail (see the cartoon in g. 3). The formation of the bow-tieshaped bundles seems to originate from an aggregation of (silica coated) fd viruses (and granular silica) after addition of the methanol/ammonia mixture, but the exact reason for the shape and size of the bundles remains unclear. We demonstrated the capability of fd viruses to be used as a template for the formation of 1D silica nanomaterials. Three nanostructures with distinct morphologies have been observed under different sol-gel conditions using TEOS as silica precursor: silica rods, wires and bow-tie-shaped bundles. Silica wires seem to transcript the bending conformation and length of intact semi-exible fd, but under somewhat different reaction conditions also remarkably straight silica rods are formed that have a high uniformity in terms of the thickness and homogeneity of the silica layer. Work devoted to further understanding the results obtained in this paper and exploring the above problems is ongoing.

[1] Z. Zhang, J. Buitenhuis, Small 2007, 3, 424. [2] Y. N. Xia, P. D. Yang, Y. G. Sun, Y. Y. Wu, B. Mayers, B. Gates, Y. D. Yin, F. Kim, Y. Q. Yan, in Advanced Materials, 2003, 15, 353. [3] G. R. Patzke, F. Krumeich, R. Nesper, Angew. Chem.Int. Edit. 2002, 41, 2446. [4] C. B. Mao, D. J. Solis, B. D. Reiss, S. T. Kottmann, R. Y. Sweeney, A. Hayhurst, G. Georgiou, B. Iverson, A. M. Belcher, Science 2004, 303, 213.

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Self-diffusion of Rod-like Viruses Through Smectic Layers.

M. P. Lettinga 1 , E. Grelet 2
1 2

IFF-7: Soft Condensed Matter Centre de Recherche Paul Pascal, CNRS-Universit Bordeaux 1, 115 Avenue Schweitzer, 33600 Pessac, France

We report the direct visualization at the scale of single particles of mass transport between smectic layers, also called permeation, in a suspension of rod-like viruses. Self-diffusion takes place preferentially in the direction normal to the smectic layers, and occurs by quasi-quantized steps of one rod length. The diffusion rate corresponds with the rate calculated from the diffusion in the nematic state with a lamellar periodic ordering potential that is obtained experimentally. Since the pioneering work of Onsager on the entropy driven phase transition to a liquid crystalline state [1], the structure and the phase behavior of complex uids containing anisotropic particles with hard core interactions has been a subject of considerable interest. Understanding of the particle mobility in the different liquid crystalline phases is more recent. In experiments various methods have been applied to obtain the ensemble averaged self-diffusion coefcients. Only a few studies have been done where dynamical phenomena are probed at the scale of a single anisotropic particle. The self-diffusion in a nematic phase formed by rod-like viruses [2] represent a recent examples, where the diffusion parallel (D ) and perpendicular (D ) to the average rod orientation (the director) has been measured. Knowledge of the dynamics at the single particle level is fundamental for understanding the physics of mesophases with spatial order like the smectic (lamellar) phase of rod-like particles. In this mesophase the particle density is periodic in one dimension parallel to the long axis of the rods, while the interparticle correlations perpendicular to this axis are short-ranged (uidlike order). For parallel diffusion to take place, the rods need to jump between adjacent smectic layers, overcoming an energy barrier known as the smectic order parameter. This process of interlayer diffusion, or permeation, was rst predicted by Helfrich [3], but has never been veried experimentally for lyotropic systems. Here we use video uorescence microscopy to monitor the dynamics of individual labeled colloidal rods in the background of a smectic mesophase formed by identical but unlabeled rods. In this way we have directly observed permeation of single rods in adjacent layers. The system of rods used in this work consists of lamentous bacteriophages fd, which are semi-rigid polyelectrolytes with a contour length of 0.88 m,

FIG. 1: (a) Overlay of DIC and uorescence images, showing smectic and two uorescently labeled particles. The cartoon shows the jump of rod-like particle between adjacent smectic layers. The layer spacing is L 0.9 m. (b) Displacement of a given particle in the direction parallel (red line) and perpendicular (black line) to the normal of the smectic layers. The green lines indicate the time for which one particle stays in a given layer.

a diameter of 6.6 nm, and a persistence length of 2.2 m. Suspensions of fd rods in aqueous solution (20 mM Tris, pH 8.2) form several lyotropic liquid crystalline phases, in particular the chiral nematic (cholesteric) phase and the smectic phase. The existence of a smectic phase in suspensions of hard rods is an evidence of the high monodispersity and therefore of the model system character of such lamentous viruses. The colloidal scale of the fd bacteriophage facilitates the imaging of individual rods by uorescence microscopy, as well as smectic layers by differential interference contrast (DIC) microscopy. Fig. 1(a) shows a sequence of images of a single region where both techniques are combined. A comparison of the images shows that some rods jump between two layers while others remain within a given layer. The trajectory of one of the rods is plotted in Fig. 1(b) in the direction parallel (z) and perpendicular (x) to the director. This gure summarizes the key observation: the diffusion throughout the smectic layers takes place in quasi-quantized steps of one rod

52

FIG. 2: The mean ordering potential in the z-direction obtained by applying the Boltzmann on the probability density function of the center of mass with respect to the average position within the layer.

FIG. 3: (a) Evolution of the self Van Hove function at dierent times. The functions are normalised to one, the z-axis is scaled by the smectic layer thickness L.

istic of a sub-diffusive behavior, while > 1 is referred to as super-diffusion. The parallel motion is close to be diffusive ( 1) in the nematic phase ( = 0.95) and reduces in the smectic phase to = 0.81, i.e. where the discrete peaks in the selfvan Hove function are observed. The perpendicular motion is in all cases strongly subdiffusive: after the nematic-smectic (N-Sm) transition it reduces from 0.68 to 0.46. This anomalous subdiffusive behavior suggests that a cage escape mechanism is at hand for both parallel and perpendicular diffusion. For parallel diffusion the cage is formed by the energy barrier imposed by the smectic layers. Perpendicular diffusion at high volume fractions is only possible through a subdiffusive reptationlike motion along the long axis to escape the local excluded volume and thus also hindered by the ordering potential. The anisotropy in the total diffusion, D /D , which is about 20 in the nematic phase [2], increases in the smectic phase. Therefore the diffusion in the smectic phase can be effectively considered as a one-dimensional diffusion of a Brownian particle in a periodic potential in the high friction limit. The diffusion coefcient in the smectic phase can then be calculated taking D0 as the diffusion coefcient in the nematic phase close to the N-Sm transition. Thus the diffusion coefcient is predicted to decrease by a factor of 0.44. Indeed, using this factor the MSD in the smectic phase is obtained from the MSD in the nematic phase. In conclusion, we have for the rst time visualized the process of permeation in the smectic phase at the scale of single particles for a system of charged rods. This allowed us to give a full and coherent description of the diffusion process without any assumptions on the system. The diffusion is strongly anisotropic in the direction normal to the smectic layers and quasi-discontinuous due to the presence of the layers. The diffusion rate complies with the rate in the nematic phase, taking into account the ordering potential, which is obtained directly from our measurements. Since the diffusion within the layer is glasslike, we conclude that the smectic phase of semi-exible hard rods consists of layers of glassy rods rather than uid layers. Thus permeation can be described in terms of Brownian particles diffusing in a one-dimensional periodic potential.

length. Moreover, it shows that the diffusion within the smectic player is extremely slow. The hoppingtype diffusion is the consequence of the underlying ordering potential imposed by the smectic layers, which can be determined experimentally from the distribution of particle positions with respect to the middle of a layer parallel to the director. The smectic ordering potential is then deduced from the Boltzmann factor for the probability of nding a particle at position z, as shown in Fig. 2. The potential can be best tted with a sinusoidal, giving an amplitude of U0 = 1.36 kB T .The use of such a potential is very common due to its simplicity [4], but this ordering potential nor its height had never been directly observed. The ordering strongly inuences the diffusion of the particles. This is exemplied by the self-van Hove function G(z, t), which is the probability for a displacement particle to a position z after a time t. Since single particles are experimentally identied, G(z, t) can be directly obtained from the histogram of particle positions after a time t, as plotted in Fig. 3. A smooth gaussian distribution that smears out over time is expected for G(z, t) for a uid made of Brownian particles. In contrast, G(z, t) shows distinct peaks exactly at integer multiples of the particle length (and therefore of the layer thickness, see Fig. 3(a)). This implies that the permeation is a function of position z, due to the ordering potential. The effect of the ordering potential is also obvious with respect to the overall mean square displacement (MSD). The time evolution of the MSD given by r2 (t) t provides the diffusion exponent : < 1 is character-

[1] L. Onsager, Ann. N.Y. Acad. Sci. 1949, 51, 627. [2] M. P. Lettinga et al., Europhys. Lett., 2005, 71, 692. [3] W. Helfrich, Phys. Rev. Lett., 1969, 23, 372. [4] B. Mulder, Phys. Rev. A, 1987, 35, 3059. [5] M.P. Lettinga and E. Grelet, Phys. Rev. Lett., 2007, 99, 197802.

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Electric Phase/ State Diagrams of Charged Fibrous Viruses (fd)

K. Kang and Jan K. G. Dhont
IFF-7: Soft Condensed Matter

We explore phase/state transitions in suspensions of charged fibrous viruses (fd) at low ionic strengths, induced by external electric fields at low frequencies where double layers are polarized. On the basis of the different observed optical morphologies, phase/state diagrams are constructed in the applied field amplitude versus frequency plane. The various phases/states and the characterization of transition lines have been investigated by pitch measurements, video correlation spectroscopy, electric birefringence and small angle dynamic light scattering. The phase/state transitions that are considered here are the result of interactions between external field-induced double layers. The phase/state diagram in the field-amplitude versus frequency plane is given in Fig.1 (see also Ref.[1]). Without an applied electric field, the equilibrium state is a nematic in coexistence with an isotropic phase. In existing literature, the nematic phase of fd-virus suspensions is found-to be chiral-nematic (in the absence of an electric field). This is the result of the chiral structure of the core of fd-virus particles. In the current study, a much lower TRIS/HCL buffer concentration is used, where the relatively large Debye length screens the chiral nature of the core of fd-particles to an extent that renders the nematic phase non-chiral. At these low buffer concentrations, the ionic strength is significantly affected by carbon dioxide that dissolves from the air (this is discussed in detail in Ref.[2]). At low applied field amplitudes (smaller than about 1.0 V/mm), coexistence between a non-chiral nematic and an isotropic state is (see the first depolarized microscopy image in Fig.1). This phase is referred to as the N-phase. On increasing the field amplitude, a chiral nematic N * -phase is induced, as can be seen from the striped patterns in the second image in Fig.1. There is a gradual transition to the ND * -phase at higher amplitudes at low frequencies, where the N-domains become smaller and disconnected and smaller sizes (see the third image in Fig.1). On further increasing the field amplitude, the chiral texture disappears and the small N-domains melt and reform. In the dynamical Ds -state, the dynamics of melting and forming is slow, while the characteristic time for melting and forming is fast (of the order of a

second) in the Df -state (where the subscripts s and f stand for slow and fast, respectively). The fourth image in Fig.1 is a snap shot of a dynamic state. At high frequencies, larger than a few kHz, the depolarized image is uniform. Birefringence measurement shown that in this H-phase the rods are homeotropically aligned [3].

E0 8 [V/mm] 6 4 2 0 0 10

[fd]=2.0 mg/ml

Df

H
Ds
* ND

N*

10

10

10 [Hz]

FIG. 1: The electric phase/state diagram of fd-virus with a concentration of 2.0 mg/ml. Several phases/states are induced at frequencies below a few kHz, while a uniform aligned phase is observed at higher frequencies..

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An electric cell is used to characterize several phases and states by various optical techniques: Electric birefringence is used to detect alignment and the degree of orientational order, especially in the H-phase. These measurements reveal that the rods are aligned along the electric field, with a degree of orientational order that is essentially independent on the field amplitude and frequency. [3]. The optical-pitch variation is measured from optical images up to the N * - ND * -transition line on variation of the field amplitude. For low amplitudes, the measured chiral -pitch shows a quite large spread, which decreases with increasing amplitude to a saturation value of about 12 m . Since the 2D projection of the optical pitch is measured, the largest measured optical -pitch is the true pitch. The large spread near the N * -

1.0

CV

[s]
15 Df ND
*

0.5
3.78

3.26

10

5 Ds

4.09 5.77

0.0

10

20 30 tim e [s]

6 7 E0 [ v/mm ]

FIG. 2: Left: Video correlation functions

ND * -transition line indicates that the director attains arbitrary values, while at higher field amplitudes the director tends to be perpendicular to the external field. The pitch is observed to
diverge at the N * - ND * -transition line. On further increasing a field amplitude at low frequency, dynamical states appear: The dynamics of melting and forming of N-domains in the Ds - and Df -states is characterized by means of video correlation functions CV , defined from the transmitted light intensity time traces as recorded by a CCD camera (a snap shot is given in the fourth image in Fig.1),
CV ( t ) =

CV for four field amplitude at a fixed low frequency (the numbers are in V/mm). The solid lines are fits to a single stretched exponent. Right: The characteristic time constant of melting and forming of small N-domains as a function of the applied field amplitude.
diverges. There thus seems to be an analogous critical behaviour as in equilibrium systems. Inter-colloidal interactions of fd-virus particles through polarized, thick electric double-layers are probably responsible for the observed phases and states. So far there is no theory that could explain the observed phase/state behaviour. It is even an open question whether polarization of condensed ions may play a role (about 85 % of the bare charge on fd-virus is screened by condensed ions). The phase/state diagram in Fig.1 is not corrected for electrode polarization. Electrode polarization arises from the formation of double layers at the electrodes that partly screen the applied electric field. In Ref.[3] we derived an expression for the attenuation factor (the ratio of the bulk-value of the electric field and the applied field amplitude). This expression shows that electrode polarization can be neglected for our electrical cell for frequencies above 100 Hz. The corrected phase/state diagrams can be obtained from this electrodepolarization theory [3].

[ I ( t ) < I ( t ) > ][ I (0) < I (0) > ] [ I (0) < I (0) > ]2

Here, I is the transmitted intensity at a given pixel and the brackets <> denote averaging over all pixels. The left plot in Fig.2 shows a few correlation functions together with fits to a singlestretched exponential. The plot on the right in Fig.2 shows the time constant for melting and forming as a function of the field amplitude. As can be seen, the time constant diverges on approach of the ND * -to- Ds transition line. Within the Df state the time constant is essentially independent of the field amplitude. Interestingly, the microscopic dynamics as probed with small angle dynamic light scattering is discontinuous at the ND * -to- Ds transition line This is probably due to the fundamentally different microstructural ordering in the ND * -phase and the Ds -state, resulting in different mobilities of the fd-viruses At the point in the phase-state diagram in Fig.1 where several transition lines meet, not only the time constant for melting and forming of N domains diverges, but also the domain size

[1] K. Kang, J.K.G. Dhont, Eur. Phys. Lett. 84, 14005, 2008 [2] K. Kang, A. Wilk, A. Patkowski, J.K.G. Dhont, J. Chem. Phys. 126, 214501, 2007 [3] K. Kang, J.K.G. Dhont, Submitted to Phys. Rev. E. 2008

55

Thermal diffusion behavior of hard sphere suspensions

H. Ning, J. Buitenhuis, S. Wiegand
IFF-7: Soft Condensed Matter

The molecular origin of the thermal diffusion process or Ludwig-Soret effect is one of the unsolved problems. It relates to our poor understanding of non-equilibrium statistical mechanics pointing out our incapability of obtaining, in some cases, even qualitative predictions, which are of practical importance in separation processes (Thermal eld ow fractionation of polymers and colloids, isotope separation), characterization of geochemical processes (Salton Sea geotherm, oil reservoir composition) and combustion. Recent advancements open up alluring perspectives to exploit thermophoresis as a novel tool in microuidic manipulation, and selective tuning of colloidal structures. One of our strategies to tackle this problem is the investigation of a spherical colloidal model system with a short range repulsive interaction potential. We studied a colloidal dispersion in the intermediate concentration range (volume fraction < 10 %) and found that the interactive part of the Soret coefcient agrees with an analytical theory. At higher concentration the Soret coefcient follows a power law. The temperature dependence of the Soret coefcient is mainly determined by single particle contributions and agrees to some extent with the temperature dependence of the surface coating material, octadecane, in toluene. Colloidal particles are small enough to exhibit thermal motion commonly referred to as Brownion motion. Being just very large molecules in a solvent, colloidal particles show many physical phenomena that are also found in ordinary molecular systems. Consequently, colloids have been used frequently to study fundamental questions in physics. Therefore, it is expected that they are also a suitable model system to illuminate the microscopic mechanism underlying the Ludwig-Soret effect, which was discovered already 150 years ago. This effect, also known as thermal diffusion, describes the diffusive mass transport induced by a temperature gradient in a multicomponent system. The scenario for a binary mixture of particles is sketched in gure 1. A number of recent studies show that interactions play an important role for the thermal diffusion behavior, where long ranged repulsion between charged micelles and colloids has been considered [1].

Conceptually, thermal diffusive behavior of highly diluted and concentrated solutions can be differentiated. In dilute solutions, where colloid-colloid interactions can be neglected, the thermal diffusion coefcient of the colloids is determined by the nature of the interactions between single colloidal particles and solvent molecules (and possibly other solutes like ions that form a double layer around the colloids) [2]. Structural changes of the surrounding solvation layer due to temperature changes and/or changes of the solvent composition may induce a sign change of the thermal diffusive behavior of single colloidal particles. Usually, the mechanism leading to a sign change is system dependent. Although, for several aqueous mixtures with and without solutes such as polymers and colloids, we found the sign change concentration is almost system independent and strongly correlated with the breakdown of the hydrogen-bond network [3]. Also the temperature dependence of ST for a large class of macromolecules and colloids in water shows a distinctive universal characteristic [1]. A pronounced concentration dependence of the Soret coefcient has been found in experiments [4, 1] and is predicted by theory [5].

Schematic illustration of the thermal diusion process in a binary mixture in a temperature gradient. The small and big particles accumulate at the hot and cold side, respectively.
FIG. 1:

In recent years, modern optical techniques have been developed which allow the investigation of complex uids with slow dynamics such as polymer solutions and blends, micellar solutions, colloidal disper-

56

sions and bio-molecules. The main issues of interest were the derivation of scaling laws and to understand the sign change of the Soret coefcient for macromolecular and colloidal systems on the basis of existing theories for molecular uids. In the past few years several theoretical concepts have been proposed to understand single particle and colloid-colloid interaction contributions to the thermophoretic motion of colloidal particles [1]. While the majority of the theoretical approaches give expressions for the single particle contribution, the work by Dhont gives explicit expressions for the contribution of colloid-colloid interactions to the thermal diffusion coefcient DT . These interaction contributions lead to a concentration dependence of the thermal diffusion coefcient. According to this theory, a sign change of the Soret coefcient as a function of temperature and concentration is possible for appropriate interaction parameters.

The measured Soret coecient () versus temperature for a colloidal suspension with a volume fraction of =10%. According to the theoretical approach by Dhont the single ( ) and the collective part ( ) of ST can be separated. The dashed lines are guides to the eye. The inset shows ST of octadecane in toluene versus temperature for a octadecane concentration of w=5 wt%.
FIG. 3:

Concentration dependence of the Soret coecient at dierent temperatures. The solid line represents a t of the data according to the theory by Dhont [5].
FIG. 2:

A separation of the Soret coefcient into a single particle contribution and an interactive part, shows, that the interactive part is almost temperature independent, while the single part shows a strong temperature dependence, which has the same tendency as the Soret coefcient of octadecane in toluene (cf. Fig. 3). But for the low molecular weight system no sign change occurs in the investigated temperature range. This might be an indication that also the silica core of the particles inuences the thermal diffusion behavior. Also the fact that octadecane is bound to a surface might inuence the thermal diffusion behavior. In general one can expect, that if the particle coating changes from " organophilic" to "organophobic" a sign change could be expected, but under which conditions this is the case needs to be claried in further investigations.

We studied spherical silica particles, sterically stabilized by octadecane, with a radius of a = 27 nm dispersed in toluene. The thermal diffusion behavior was studied in a concentration range between 1% and 30% by volume fraction and in a temperature range from 15-50 C [6]. Fig. 2 shows the Soret coefcient as function of the volume fraction for different temperatures. At low temperature the colloids move to the warm side, while at high temperatures the colloids move to the cold side. For intermediate temperatures a sign change of the Soret coefcient occurs for higher concentration. Typically, the movement of the solute particles to the warm side is an indication for poor solvent conditions, while under good solvent conditions the particles move to the cold side. For the investigated system the vicinty of a gel line at low temperatures is probably responsible for movement of the colloids to the warm side. In the intermediate concentration range the concentration dependence of the Soret coefcient can be described by the theory for hard spheres [5]. At high concentrations ST follows a power law as it also has been found for polymers approaching the glass-transition [7]. Although the exponent is two orders of magnitude smaller.

[1] R. Piazza. A. Parola, J. Phys-Cond. Matt 20, 153102 (2008). [2] J.K.G. Dhont, Eur. Phys. J. E 25, 61 (2008). [3] S. Wiegand, H. Ning, and R. Kita, J. Non-Equilib. Thermodyn., 32, 193 (2007). [4] H. Ning, R. Kita, H. Kriegs, J. Luettmer-Strathmann, and S. Wiegand, J. Phys. Chem. B. 110, 10746 (2006). [5] J. K. G. Dhont, J. Chem. Phys. 120, 1632 (2004) and 1642 (2004). [6] H. Ning, J. Buitenhuis, J. K. G. Dhont, and S. Wiegand, J. Chem. Phys. 125, 204911 (2006). [7] J. Rauch and W. Khler, Macromolecules 38, 3571 (2005).

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58

ISB-1: Cellular Biophysics

Acting Director: Prof. Frank Mller

The Institute of Structural Biology and Biophysics, Cellular Biophysics ISB-1 (formerly INB-1 or IBI-1) is dedicated to research in molecular and cellular neurobiology, signal transduction, information processing, and cell bio-physics. The cell is the smallest living unit. In every cell, chemical and physical stimuli evoke characteristic physiological responses. Signal transduction usually begins with receptor proteins that register a stimulus and start a cascade of biochemical reactions which often ends with the opening or closure of ion channels that changes the electrical properties of the cell membrane. Moreover, in order to adapt to changes in the environment each cell must constantly modulate these signalling processes. In ISB-1, signal transduction and information processing are studied at different levels of complexity: on the molecular level we study the properties of receptor proteins and ion channels. On the cellular level, G-Protein-mediated signalling as well as mechanisms of excitation and adaptation of sensory cells and neurons are investigated. On the network level, we study information processing in small well-characterized neuronal circuits, such as the retina or the olfactory bulb.

A strength of the ISB-1 has always been the tight co-operation of biologists, chemists, and physicists. Our technical repertoire reaches from the analysis of proteins using molecular biological and biochemical methods to physiological studies in vitro and in vivo in normal and in transgenic animals. Fluorescence-based optical methods and imaging play an important role in the research of ISB-1. To monitor the concentration of intracellular messengers like calcium or cAMP, imaging techniques have been estab-lished, including twophoton fluorescence-lifetime imaging microscopy, single molecule fluorescence spectroscopy, and camera-based ratiometric imaging setups. Both synthetic fluorescent dyes as well as genetically encoded sensors based on fluorescent proteins are employed in the institute. The analysis of cellular processes with high spatial and temporal resolution provides the basis for an in-depth understanding of the physics of the cell.

59

The Role of HCN4 channels in Cardiac Pacemaking

D. Harzheim1, R. Seifert1, K.H. Pfeiffer1, L. Fabritz2, E. Kremmer3, T. Buch4, A. Waisman5, P. Kirchhof2, U. B. Kaupp1
1 3

ISB-1: Cellular Biophysics; 2Med. Klin. & Poliklinik,Univ. Mnster und IZKF Mnster, D-48129 Mnster Helmholtz Zentrum Mnchen, D-81377 Mnchen; 4Exp. Immunologie, Univ. Zrich, CH-8057 Zrich 5 I. Med. und Poliklinik, Univ. Mainz, D-55131 Mainz Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels belong to the superfamily of voltage-gated ion channels. The depolarizing current flowing through HCN channels, designated as Ih or If, plays an important role in controlling cardiac rhythmicity. Moreover, HCN channels are important targets for adrenergic stimulation. Upon activation of adrenergic receptors, cAMP levels increase, leading to a higher open probability of HCN4 channels. We studied the role of If in mice, in which binding of cAMP to HCN4 channels was abolished by a single amino-acid exchange (R669Q) [1]. Homozygous R669Q/R669Q mice die during embryonic HCN4 development. Prior to E12, homozygous and heterozygous embryos display reduced heart rates and show no or attenuated responses to catecholaminergic stimulation. Adult heterozygous mice display normal heart rates at rest and during exercise. However, following adrenergic stimulation, hearts exhibit pauses and sino-atrial node block. These results suggest that in the embryo, HCN4 is the principal cardiac pacemaker and persistent elevation of the heart rate by cAMP is essential for viability. In adult mice, HCN4 channels take no longer part in heart rate regulation, but prevent sinus pauses during and after stress. Thus, the mechanism of pacemaking may switch during development and HCN4 may serve two different functions that critically rely on the presence of cAMP. Spontaneous activity of the mammalian heart is generated in the sino-atrial node (SAN). The question how pacemaker activity is generated in SAN cells and how pacemaking is regulated by the autonomouos nervous system is still a matter of debate. Hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels are thought to play a major role in cardiac pacemaking. Within the superfamily of voltage-gated channels, HCN channels are unique in that they are activated upon hyperpolarization rather than by depolarization of the membrane potential. In addition, voltagedependent opening of these channels is regulated by the direct binding of cAMP to the polypeptide. Native HCN channels consist of four subunits. In mammals four genes (HCN 1 4) have been identified to encode individual subunits. The If current, mediated by HCN channels, is activated at negative membrane potentials. If leads to excitation of the cell and spontaneous firing of action potentials and is, therefore, thought to be one of the primary ionic mechanisms generating cardiac spontaneous rhythmic activity. In this study, we decided to investigate the precise role of HCN4 in cardiac pacemaking of mice. We generated knock-in mice that harbour a single amino-acid exchange (R669Q) in the cyclic nucleotide-binding domain of the HCN4 channel that abolishes cAMP binding to the protein. The arginine residue is crucial for the binding of cAMP, because it interacts with the negatively charged phosphate group. To ascertain that the mutation abolishes regulation by cAMP, we analyzed the wild-type and the HCN4R669Q mutant after heterologous expression in a cell line. The voltagedependent activation of HCN4R669Q mutant and wild-type channels is similar. Cyclic AMP shifted the activation curve of the wild-type HCN4 channel by +25 mV, whereas no shift was observed for the HCN4R669Q mutant (Fig. 1).

Fig. 1: Electrophysiological characterization of HCN4 and R669Q HCN4 channels. Voltage-dependent activation of R669Q heterologously expressed HCN4 (circles) and HCN4 channels (squares). Voltages of half-maximal activation ( SD) in the absence (open symbols) and presence (filled symbols) of cAMP (100 M) were -79.3 3.7 (n = 8) and 55.6 2.7 mV (n = 3) for HCN4, and -84.5 3.4 (n = 7) R669Q and -81.3 7.1 mV (n = 5) for HCN4 , respectively.

We analyzed the spontaneous beat frequency of +/+ +/R669Q hearts isolated from HCN4 , HCN4 , and

60

HCN4R669Q/R669Q embryos prior to E11.5. Under basal conditions, hearts from heterozygous and homozygous embryos beat regularly without obvious arrhythmias; however, the heart rate was significantly slower compared to hearts from wildtype embryos (Fig. 2). Furthermore, in wild-type embryos, the heart rate increased during E9 to E11 of embryonic development (Fig. 2). This increase was no longer present in HCN4+/R669Q embryos R669Q/R669Q embryos, the heart rate whereas in HCN4 is significantly reduced (Fig. 2). Notably, the beat frequency of hearts from HCN4R669Q/R669Q and HCN4-/- embryos at E9.5 was identical, demonstrating that the point mutation in the HCN4 channel has the same effect on the basal heart rate as the complete loss of the channel [2].

perfused hearts, mostly during washout of the adrenoceptor agonist orciprenaline (Fig. 3; pauses in 4/10 HCN4+/R669Q hearts vs. 1/9 HCN4+/+ hearts; during orciprenaline infusion: pauses in 5/10 HCN4+/R669Q vs. 1/9 HCN4+/+ hearts; during washout of orciprenaline: pauses in 8/10 HCN4+/R669Q vs. 0/9 +/+ HCN4 hearts).

channel alters the embryonic heart Fig. 2: The HCN4 beat. Basal heart rate during embryonic development +/+ +/R669Q (E9.5-11, HCN4 : red, HCN4 : green, R669Q/R669Q : blue). The number of embryos analyzed HCN4 is indicated.

R669Q

Isoproterenol superfusion increased the rate of isolated hearts from HCN4+/+ and HCN4+/R669Q embryos (~ 37.4 % and 19.8 %, respectively), whereas no increase was observed in hearts from HCN4R669Q/R669Q embryos (2.8 %). Similarly, superfusion with NKH477, an activator of membrane-bound adenylyl cyclases, increased the heart rate from HCN4+/+ and HCN4+/R669Q embryos, R669Q/R669Q embryos. These but not from HCN4 results demonstrate that HCN4 is the principal target for cAMP during the embryonic stages of mouse development. The differences in basal heart rate and in the activation of If between wild-type and heterozygous embryos prompted us to study the heart rate in freely moving adult HCN4+/+ and HCN4+/R669Q mice by telemetric recording of ECG. Neither the heart rate at rest nor during exercise or mental stress was different between genotypes. The heart rate of sedated animals and the intrinsic rate of the isolated heart was also not different between genotypes, nor was the electrophysiology of the atrio-ventricular node and the ventricle. After +/R669Q mice developed pauses and exercise, HCN4 sino-atrial block more often than their wild-type littermates (Fig. 3, HCN4+/R669Q: 9 7 pauses per +/+ 55 minutes, HCN4 : 6.5 5 pauses per 55 minutes). Sino-atrial block and sinus pauses were also found in spontaneously beating, Langendorff-

Fig. 3: HCN mice display a sino-atrial block. a, Representative examples of telemetric ECG recordings in freely moving mice carrying an implanted ECG transmitter. All recordings were obtained during the 55 minute recovery period after stress tests (air jets or swimming). Animal numbers are indicated. b, Representative recording of spontaneous rhythm in isolated, Langendorffperfused hearts. Shown is a ventricular monophasic action potential (MAP), a right atrial electrogram (RA), and lead II of the tissue bath ECG (ECG). The WT heart shows a +/R669Q constant sinus rhythm, the HCN4 heart shows three pauses with doubling of cycle length, consistent with sinoatrial block.

+/R669Q

Our results show that only in embryonic mice, HCN4 channels serve as pacemaker controlling the heart beating frequency. Notably, this activity is severely affected when cAMP modulation of the channel is impaired. In adult mice, however, the contribution of HCN4 channels is less pronounced. Here, the channels may serve a back-up mechanism that maintains a stable heart beat in situations during and after stress and, HCN4 channels are probably no longer involved in sympathetic stimulation of the heart rate.

[1] D. Harzheim, K.H. Pfeiffer, L. Fabritz, E. Kremmer, T. Buch, A. Waisman, P.Kirchhof, U.B. Kaupp and R. Seifert, EMBO J. 27, 692 (2008). [2] J. Stieber, S. Herrmann, S. Feil, J. Lster, R. Feil, M. Biel, F. Hofmann and A. Ludwig, Proc. Natl. Acad. Sci. USA 100, 15235 (2003)

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Light Responses in the Mouse Retina are Prolonged Upon Targeted Deletion of the HCN1 Channel Gene
F. Mller1, G.Knop1, M.W. Seeliger2, F. Thiel1, A. Mataruga1, U.B. Kaupp1, C. Friedburg3, N. Tanimoto2
1 2

ISB-1: Cellular Biophysics Inst. Ophthalmic Res. Univ. Tbingen, D-72076 Tbingen 3 Dept. Ophthalmology, Univ. Giessen and Marburg, D-35390 Giessen

In the mammalian retina, the light response of photoreceptors is processed by an elaborate neuronal network. The photoreceptors, rods and cones, are depolarized in the dark, but hyperpolarize upon illumination. At least ten types of cone bipolar cells and one type of rod bipolar cell provide the pathways for the signal flow from photoreceptors to ganglion cells, the output neurons of the retina. Along the way from photoreceptors to ganglion cells, cellular signals are shaped by two principal mechanisms: 1. Lateral inhibition within the neuronal network involving feedforward and feedback mechanisms and 2. Shaping of voltage responses by the kinetic properties of the ion channels involved in the generation and propagation of electrical signals. One family of voltage-gated channels present in the retina are the HCN channels, which are activated by hyperpolarization and gated by cyclic nucleotides. In mammals, four channel genes (HCN1-4) have been identified. Here, we show that HCN1 is particularly strong expressed in both rod and cone photoreceptors. By recording scotopic and photopic light responses in normal mice and in HCN1 knock-out mice, we show that HCN1 channels shorten retinal light responses in both rod and cone pathways [1]. HCN channels co-determine the resting potential and membrane conductance and, thereby, play an important role in the integrative behaviour of neurons and the sensitivity to synaptic input. HCN channels affect the cable properties of dendrites and shape the time course and propagation of excitatory and inhibitory postsynaptic potentials. We have previously shown that HCN channel isoforms are differentially expressed in the retina [2, 3]. In rod and cone photoreceptors, HCN channels were suggested to shape the light response. HCN channels become activated during hyperpolarization in bright light and depolarize the cell toward the dark membrane potential, making the light response transient.

HCN1 is strongly expressed in the mouse retina HCN1 is expressed throughout the retina. In the outer retina, rod photoreceptors are strongly HCN1-immunoreactive. Cone photoreceptors that can be labelled with antibodies against the calcium-binding protein CabP5 are also positive for HCN1.

Fig. 1: Immunohistochemical localization of HCN1 in the mouse retina. (A) HCN1-specific antibody labels photoreceptor inner segments, somata, axons and axon terminals in the outer retina. Somata of certain bipolar cells (long arrow) and amacrine cells (short arrow) are labelled in the INL, corresponding bands of bipolar cell axon terminals (long horizontal arrow) and amacrine cell dendrites (arrowhead) are visible in the IPL. Some ganglion cell somata are also HCN1-positive in the GCL. (B) No HCN1 immunoreactivity is observed in retinal sections obtained from HCN1 knock-out animals. (C) CabP5-immunoreactivity is detected in a subset of cone photoreceptors. (D) Double staining against CabP5 (left) and HCN1 (right) shows that HCN1 is present in the plasma membrane of cones (arrow). OS, outer segments; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Light responses are prolonged in HCN1 knock-out mice The murine Ganzfeld ERG is a measure of the overall retinal function. We performed single-flash ERG recordings under scotopic and photopic adaptation conditions and with different stimulus intensities. Wild-type and HCN1 knock-out animals were littermates (six weeks of age). Representative records of a wild-type mouse are

62

shown in the left columns of Fig. 2A (scotopic) and B (photopic). Typically, ERG waveforms consist of an early negative-going component best visible at high light intensities, the a-wave, followed by a large positive-going component, the b-wave. Whereas at least the initial portion of the a-wave reflects the primary light response in photoreceptors, the b-wave is dominated by the activity of ON-bipolar cells. Riding on top of the bwave are the oscillatory potentials, small wavelets that probably involve inner retinal circuitry. In the scotopic regime, the knock-out of HCN1 channels had no substantial effect on the onset of the ERG response or the amplitudes of the a- and b-waves (Fig. 2A, C). In the statistical analysis (Fig. 2D, n= 4 for both wild-type and HCN1 knock-out animals), the scotopic b-wave amplitude data recorded from HCN1 knock-out animals (box plots) fall well within the black lines that limit the 5% to 95% normal range of the wild-type data. In contrast, the photopic (light-adapted) responses are reduced and their oscillatory components also diminished (Fig. 2B,C). The most prominent effect of HCN1 knock-out concerned the duration of light responses. Under both scotopic and photopic conditions, the b-wave was considerably prolonged in HCN1 knock-out animals at higher light intensities.

Flicker detection is compromized in HCN1 knockout mice As single flash responses are prolonged, it is tempting to speculate that already at the level of the retina the flicker fusion frequency, i.e. the ability to respond to repetitive stimulation, should be reduced. The following figure compares ERG recordings in wildtype and HCN1 knock-out animals to repetitive light stimuli at different frequencies and different light intensities. At low flash intensities, light responses in wildtype and knock-out animals were similar, presumably because the hyperpolarization of the photoreceptors was too small to activate HCN1 channels. Indeed, at 10 mcd*s/m, the flicker data were rather similar. In contrast, at higher flash intensities like 300 mcd*s/m that caused a manifest prolongation of the responses, there was a remarkable reduction of the ability to follow higher frequency flicker in the knock-out animals.

Fig. 3: Effect of HCN1-deficiency on scotopic ERG flicker fusion frequency. (A) At low flash intensities like 0.01 cd*s/m, there were no substantial differences between wt and knock-out mice. (B) At higher flash intensities like 0.3 cd*s/m, the apparent prolongation of the waveform led to a marked reduction in the ability to follow high frequency stimulation.

Our results show that already in the first cell of our visual system, i.e. the photoreceptor, light responses are significantly modulated.

Fig. 2: Effect of HCN1-deficiency on retinal function under scotopic and photopic conditions. Direct comparison of flash ERG recordings in dark-adapted (A) and light-adapted (B) wild-type (Wt) and knock-out mice -/(HCN1 ). (C) Superposition of recordings from wild-type and knock-out animals reveals that light responses are considerably prolonged in knock-out mice. (D) Statistical evaluation of the corresponding ERG b-wave data. Boxes indicate 25 % and 75 % quantile range, whiskers the 5 % and 95 % quantiles and the asteriks the median -/of the HCN1 data. The normal range is delimited by black lines indicating the 5 % and 95 % quantile of the wild-type data.

[1] G.C. Knop, M.W. Seeliger, F. Thiel, A. Mataruga, U.B. Kaupp, C. Friedburg, N. Tanimoto and F. Mller, Eur. J. Neurosci. 28, 2221 (2008) [2] E. Ivanova and F. Mller, Vis. Neurosci. 23, 143 (2006). [3] F. Mller, A. Scholten, E. Ivanova, S. Haverkamp, E. Kremmer and U.B. Kaupp, Eur. J. Neurosci. 17, 2084 (2003).

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Molecular and Functional Properties of Native and Heterologously Expressed Adenylyl Cyclases
A. Baumann1, S. Wachten1, N. Fuss1, R. Gauss1, J. Schlenstedt2, S. Mujagic3, J. Erber3
1 2

ISB-1: Cellular Biophysics Institute of Biochemistry and Biology, Univ. Potsdam, D-14476 Golm 3 Institute of Ecology, TU Berlin, D-10587 Berlin

Cyclic AMP serves as an important intracellular messenger in virtually all organisms. This small organic compound participates e.g. in sensory signal transduction, cardiac myocyte regulation, as well as in learning and memory. Production of cAMP is based on the activity of adenylyl cyclases (ACs). A variety of factors can modulate the properties of these enzymes and lead to dynamic changes of the intracellular cAMP concentration. Recently, we cloned the first gene from the honeybee (Apis mellifera) encoding a membrane-bound AC (Amac3) [1]. Molecularly, this gene is orthologous to the mammalian olfactory AC type 3. The enzymatic properties of AmAC3 were determined after heterologous expression of the gene in a cell line. We have now compared the biochemical and pharmacological properties of heterologously expressed AmAC3 with native ACactivity in identified subregions of the honeybee brain [2]. Values for half-maximal activation with a water-soluble analogue of forskolin (NKH477) of both, cloned and native ACs, were in the low micromolar range. Biosynthesis of cAMP was specifically blocked by P-site inhibitors. The same rank order of inhibitory potency was shared between AmAC3 and ACs in the antennal lobes of honeybee brain. Our results suggest a role for AmAC3 in sensory, motor and higher-order information processing in the honeybee brain. Membrane-bound ACs (tmACs) share a common and characteristic topology with two large hydrophobic membrane domains (M1 and M2) within which the amino-acid chain spans the plasma membrane six times. The catalytic domains of the enzyme reside in the cytosolic loop connecting the M1 and M2 domains and in the cytoplasmic C-terminus, following the M2 domain. All tmACs are activated by subunits of stimulatory G-proteins (Gs). Most tmACs are also activated by the diterpene forskolin or NKH477 (s.a. [3]). As a result of AC-activity, the concentration of the intracellular messenger cAMP increases. This messenger regulates and modulates the activity of protein kinases, ion

channels as well as transcription factors. Therefore, changes of the cAMP concentration can have a significant impact on cellular signaling behavior. After cloning the Amac3 gene from honeybee brain, we performed a phylogenetic analysis of the derived amino acid sequence to a variety of vertebrate and invertebrate AC sequences. A dendrogram was constructed (Fig. 1) that showed the closest relationship between AmAC3 and AC sequences from rat olfactory neurons (RnAC3; [4]) and the Drosophila protein (DmAC39E; [5]).

Fig. 1: Phylogenetic comparison of AmAC3 with mammalian, Drosophila, and C. elegans ACs. The amino-acid sequences were obtained from NCBI databases RnAC1 , AC1 from Rattus norvegicus (Rn); MmAC2, AC2 from Mus musculus (Mm); RnAC3; RnAC4; RnAC5; RnAC6; MmAC7; MmAC8; MmAC9; DmACrutabaga; AC1 from Drosophila melanogaster (Dm); DmAC78C; DmAC76E; DmAC35C; DmAC39E); DmAC10B; CeACY1; AC1 from Caenorhabditis elegans (Ce); CeACY2; CeACY3; CeACY4. The sequence of the soluble MmAC10 was used as out-group. The numbers at the nodes represent the per cent bootstrap support for each branching. The scale bar allows the conversion of branch lengths in the dendrogram to genetic distance between clades (0.1 = 10% genetic distance).

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In order to determine the enzymatic properties of AmAC3, a cell line was generated (Fig. 2) that constitutively expresses AmAC3 and a cAMPgated ion channel (CNG). This CNG channel is permeable to Ca2+ ions. Therefore, an increase in 2+ AmAC3 activity can be monitored by Ca -imaging due to the subsequent activation of the CNG channel. We used this cell line as well as a cell line that only expresses the CNG channel to determine the dose-response curve of AmAC3activation by forskolin, a specific agonist of tm-ACs (Fig. 3). In addition, the cell lines were used to monitor the G-protein coupled receptor mediated activation of AmAC3. In both cell lines adrenergic receptors are expressed endogenously. The EC50 values for activation of AmAC3 by forskolin and norepinephrine were 15.2 M and 3.1 M (Fig. 3).

Fig. 3: Effects of AC activation on Ca -dependent Fluo-4 signals in AmAC3-expressing (flpAC3) and parental (flpTM) cells. Data are mean values of two independent experiments performed in duplicate. Error bars indicate the standard deviation. Relative fluorescence intensities are given in counts / mg total protein. Above: Fluo-4 signals of flpTM- and flpAC3 cells evoked by AC stimulation with increasing forskolin concentrations (0.1150 M). Below: Fluo-4 signals in flpTM- and flpAC3 cells evoked by AC stimulation with increasing norepinephrine concentrations (0.01-30 M).

2+

15 m

The distribution of Amac3 transcripts and protein in honeybee brain was examined by in situ hybridization and immunological staining [1, 2]. Using protein preparations of subregions of the honeybee brain and from the AmAC3-expressing cell line, we analysed the biochemical and pharmacological properties of native and cloned enzymes. Adenylyl cyclases expressed in the antennal lobes of honeybees and AmAC3 share very similar, if not identical properties. These results have been submitted for publication recently [2]. The prominent expression of AmAC3 in the antennal lobes of honeybee brain suggests AmAC3 to participate in the processing of olfactory signals. Therefore, AmAC3 may serve in vivo to translate GPCR- and/or Ca2+-mediated signals into changes of the intracellular cAMP concentration and thus to participate in sensory, motor and even higher brain functions.

Fig. 2: Immunocytochemical detection of heterologous expressed AmAC3. HA-tagged AmAc3 was visualized using a rat anti-HA-antibody and a fluorescently labeled secondary antibody (goat-anti-rat ALEXA 568). (Left) Light-microscopic photograph of a cell. (Right) The same cell viewed under fluorescence-microscopic conditions. The AmAC3 protein is located in the plasma membrane (arrows).

[1] S. Wachten, J. Schlenstedt, R. Gauss and A. Baumann, J. Neurochem. 96, 1580 (2006). [2] N. Fuss, S. Mujagic, J. Erber, S. Wachten and A. Baumann, submitted. [3] Y. Chern, Cell. Signal. 12, 195 (2000).

rel. Fluorescence [counts / mg total protein]

8x106

flpTM flpAC3

[4] H.A. Bakalyar and R.R. Reed, Science, 250, 1403 (1990). [5] V. Iourgenko and L.R. Levin, Biochim. Biophys. Acta, 1495, 125 (2000).

6x106

4x106

2x106

0,1

10 Forskolin [M]

100

rel. Fluorescence [counts / mg total protein]

8x106 flpTM flpAC3 6x106

4x106

2x106

0.01

0.1

1 Norepinephrine [M]

10

100

65

Functional Studies of a Prokaryotic Cyclic Nucleotide-Gated Channel

A. Cukkemane, B. Grter, K. Novak, T. Gensch, W. Bnigk, R. Seifert, U. B. Kaupp
ISB-1: Cellular Biophysics Cyclic nucleotide-gated (CNG) channels play a fundamental role in signal transduction of sensory neurons. Upon binding of cyclic nucleotides, CNG channels open and thereby cause changes in membrane potential. Although the physiological role of CNG channels has been extensively studied, the molecular events that relay ligand binding to channel activation are not well understood. The molecular interactions that tune affinity and selectivity of cyclic nucleotide binding are not fully understood, because ligand affinity has not been measured directly but rather has been inferred from electrophysiological studies. As binding and gating are intimately coupled, it is difficult to separate one from the other. Here, we examined the ligand-binding properties of a K+-selective CNG channel that was cloned from the bacterium Mesorhizobium loti (mlCNG) [1]. Ligand binding studies were performed on heterologously expressed and purified tetrameric mlCNG protein as well as on its isolated cyclic nucleotide-binding domain (CNBD). Both, the mlCNG protein and the CNBD bind cAMP in a non-cooperative manner with similar affinity. These results indicate that the binding and gating properties of the bacterial channel are distinctively different from those of mammalian CNG channels. In order to perform binding studies on purified protein, the mlCNG was expressed as a fusion protein with a hexa-histidine tag at its C-terminal end. This allowed purifying large amounts of the 2+ protein using a Co affinity column. A fluorescent cAMP-analogue, i.e. 8-NBD-cAMP, was used to study ligand binding to mlCNG. The NBD-group is a polarity sensitive molecule. It is largely non-fluorescent in aqueous solution and becomes fluorescent in a hydrophobic environment. This property of the NBD group has been widely utilized in biophysical studies on lipids as well as on proteins, like epithelial exchange factor (EPAC). We observed a pronounced fluorescence signal upon binding of 8-NBD-cAMP to purified mlCNG. This allowed determining quantitatively the binding affinity of the channel for this ligand (Fig. 1A). The data were analyzed assuming a simple binding model. The mean KD value for 8-NBD-cAMP was 15.9 2.7 nM. To determine the KD values of physiological cyclic nucleotides, i.e. cAMP and cGMP, the binding of 8-NBD-cAMP to the mlCNG protein was competed with non-labelled cyclic nucleotides (Fig. 1B). The KD value was 81.6 17.5 nM for cAMP and 320.7 25.5 nM for cGMP. Next we determined whether the binding properties of the tetrameric channel differed from the isolated CNBD. Previously, the wildtype CNBD and a mutated version (R348A), that has a much lower affinity, have been expressed and purified as GST-fusion proteins to determine their crystal structures either in the ligand-bound or ligand-free (R348A) state [2]. We expressed both variants of the CNBD and purified the proteins on a glutathione affinity column. The presence of a thrombin cleavage site allowed us, to purify the CNBD without its GST fusion partner. We observed that cAMP co-purifies with wildtype CNBD. In contrast, R348A has no cAMP bound. To perform ligand binding experiments with the wildtype CNBD, it was necessary to unfold and refold the CNBD to obtain cAMP-free protein.

Fig. 1: Ligand binding of the full-length mlCNG protein. (A) Normalized increase of fluorescence of 8-NBD-cAMP on binding to mlCNG protein (0.5 M). 8-NBD-cAMP fluorescence in the absence of the mlCNG protein was subtracted. The solid line represents non-linear leastsquares fit. The KD value was 17.3 nM. (B) Competition between 8-NBD-cAMP (0.5 M) and cAMP (closed circles) or cGMP (open circles) for binding to the mlCNG protein (0.3 M). The solid lines represent non-linear least-squares fit. The KD values were 71.3 nM (cAMP) and 363.9 nM (cGMP).

Ligand binding to purified CNBDs was studied using the same assay as for the intact channel (Fig. 2). The KD values were 67.8 8.7 nM for cAMP and 300.4 15.4 nM for cGMP for the wildtype CNBD. The KD values obtained for the R348A mutant were 18.5 4.3 M for cAMP, and 22.3 5.7 M for cGMP.

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residue is also a crucial determinant of cNMP-binding to the CNBD of the hyperpolarization-activated and cyclic nucleotidegated (HCN) 2 channel [5]. The arginine residue interacts directly with cAMP. The CNBD in CNG channels is lacking this particular residue. This indicates that the arginine residue may be one of the determinants of ligand selectivity and sensitivity. The KD values derived for R348A are in the micromolar range, i.e. ~ 400 fold lower than for the wild-type CNBD or mlCNG channel. Interestingly, the mutated CNBD has similar binding affinities for cAMP and cGMP. These results clearly indicate that both, ligand selectivity and sensitivity are affected by this single amino acid exchange and thereby foster the molecular understanding of the CNG channel activation process.
Fig. 2: Ligand binding to the CNBD protein. (A) Increase of 8-NBD-cAMP fluorescence on binding to the CNBD protein (0.5 M). 8-NBD-cAMP fluorescence in the absence of the CNBD protein was subtracted. The solid line represents nonlinear least-squares fit. The KD value was 17.6 nM. Inset: emission spectrum of 8-NBD-cAMP (1 M) in the absence (black) and presence of CNBD protein (1 M) (red). (B) Competition between cAMP (closed circles) or cGMP (open circles) and 8-NBDcAMP (1 M) for binding to CNBD (1 M). The KD values were 73.5 nM (cAMP) and 296.9 nM (cGMP). (C) Increase of fluorescence of 8-NBD-cAMP (0.5 M) on binding to increasing concentrations of the mutant CNBD (R348A). The KD value was 7.3 M. (D) Competition between cAMP and cGMP with 8-NBD-cAMP for binding to mutant CNBD (R348A) (3 M). The KD values were 22.9 M (cAMP) and 27.7 M (cGMP).

[1] A. Cukkemane, B. Grueter, K. Novak, T. Gensch, W. Boenigk, R. Seifert, U. B. Kaupp EMBO Rep. 8, 749 (2007). [2] G.M. Clayton, W.R. Silverman, L. Heginbotham and J.H. Morais-Cabral, Cell 119, 615 (2004). [3] C.M. Nimigean, T. Shane and C. Miller, J. Gen. Physiol. 124, 203 (2004). [4] U.B. Kaupp and R. Seifert, Physiol. Rev. 82, 769 (2002). [5] W.N. Zagotta, N.B. Olivier, K.D. Black, E.C. Young, R. Olson and E. Gouaux, Nature 425, 200 (2003).

These results show that the binding affinity is very similar for the tetrameric channel and the wildtype CNBD. However, the affinity for cAMP-binding is 4 to 5-fold higher than for cGMP-binding suggesting that physiologically, mlCNG represents a cAMPgated channel. Comparing our results obtained from ligand-binding experiments with those of channel activation [3] reveal that the KD of cAMP-binding coincides with the K1/2 of channel activation by cAMP. The KD of cGMP binding is 2 fold lower than K1/2 of cGMP-dependent channel activation. These findings strongly suggest that binding and gating in the mlCNG channel is a noncooperative process. A corollary is that the binding event is not affected by intersubunit contact and that binding sites in the tetrameric channel act independently of each other. In contrast to mlCNG, activation of CNG channels expressed in photoreceptors or olfactory neurons is a cooperative process. Although we could determine the KD values for cAMP and cGMP binding to the mlCNG channel in quantitative terms, the molecular basis that determines ligand affinity and ligand selectivity of CNG channels still is not well understood. Several residues in the CNBD as well as at sites distant from the CNBD affect cyclic nucleotide binding [4]. We observed a pronounced change of the binding affinity in the mutated CNBD (R348A). The mutation of this arginine residue results in a CNBD that could be purified in cAMP-free form. This

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Parallel Information Processing Starts at the First Synapse in the Visual System
F. Mller1, E. Ivanova1, A. Mataruga1, C. Puller2, S. Haverkamp2, H. Wssle2
1 2

ISB-1: Cellular Biophysics MPI Brain Research, D-60528 Frankfurt

In the mammalian retina, the light response of rod and cone photoreceptors is processed by an elaborate neuronal network (for review see [1], [2]). At least ten cone bipolar cell types and one rod bipolar cell type provide the direct pathways from the photoreceptors to the ganglion cells that relay the information to the brain. Most importantly, these bipolar cell types form the basis for parallel processing of visual signals. We, therefore, investigated the connectivity and the functional properties of all bipolar cell types. Each cone feeds its signal into every bipolar cell type. Patch-clamp recordings revealed that each morphologically identified bipolar cell type expresses a unique inventory of voltage-gated ion channels. These results suggest that bipolar cell types differ in their functional properties, each one being specialized to filter certain aspects from the visual information. Finally, we identified bipolar cells that provide for the recently proposed alternative rod pathway. Bipolar cells establish parallel pathways In the mammalian retina the bipolar cells provide parallel pathways to relay the information from the photoreceptors to the ganglion cells. Cones feed into ON- and OFF-cone bipolar cells that excite ON- and OFF-ganglion cells, respectively. In the classical rod pathway, rods feed into rod bipolar cells that provide input into both the ON- and the OFF-pathway via AII amacrine cells. In a collaboration with the MPI for Brain Research in Frankfurt, we extended our studies on the identification of bipolar cell marker proteins, bipolar cell densities, connectivity to photoreceptors, and bipolar cell axonal tiling in the mouse retina. We found that the dendritic trees and axon terminals of each bipolar cell type tile the retina without much overlap. Each and all cones are connected to at least one member of any given type of cone bipolar cell [3]. In a highly complex synaptic formation, the cone pedicle provides output onto at least ten different postsynaptic bipolar cells. Thus, parallel information processing starts at the very first synapse in the visual system.

Each bipolar cell type expresses a unique inventory of ion channels The light response of bipolar cells is shaped by two different means: by the precise timing of excitatory and inhibitory input and by the kinetics of the receptors and ion channels involved in the generation of the input and in the propagation of the electrical signal. Using the patch-clamp technique in the whole cell configuration we studied the expression of voltage-gated ion channels in bipolar cells in rat retinal slices. While recording, cells were filled with Lucifer yellow to reveal their morphology. Figure 1 shows all bipolar cell types revealed in our study. Note that the axons of different bipolar cell types stratify at different levels of the inner plexiform layer (IPL). The traces show representative current recordings of type 4, type 5, and type 6a bipolar cells. From the holding potential of -60 mV, the membrane potential was clamped to values from -65 to -125 mV in -15 mV increments in a family of 500 ms long hyperpolarizing voltage steps. In type 5 bipolar cells, these voltage steps elicit pronounced slowly activating inward currents (seen as downward deflections). Upon depolarization to 0 mV, large sustained outward currents are measured (seen as upward deflection at the end of the trace). These membrane currents are conducted by different ion channel types. For example, the inward currents are carried by hyperpolarizationactivated and cyclic nucleotide-gated channels (HCN channels), while outward currents are conducted by outwardly rectifying potassium channels. Current traces recorded from other bipolar cell types look different, indicating differences in the expression of ion channels. For example, type 4 bipolar cells show no inward currents and, therefore, do not express HCN channels. Type 6a bipolar cells show inward currents; however, their shape differs from those found in type 5 bipolar cells. In type 6a bipolar cells, currents carried by calcium-activated chloride channels are superimposed on HCN channel currents. We found that each morphologically identified bipolar cell type expresses a unique inventory of voltage-gated ion channels suggesting that different bipolar cell types differ in their functional properties. The

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different repertoires of currents can serve as finger prints to identify the bipolar cell types. Moreover, our combined electrophysiological and morphological approach allowed us to describe a new type of rat ON-cone bipolar cell (type 6b) that, based on morphological criteria alone, would not have been classified as a distinct cell type [4].

Fig. 2: Electronmicrograph. Dendrites of one type of OFF-cone bipolar cell labeled by antibodies against protein kinase A (arrows) form basal contacts at a rod spherule (rs). M, mitochondrium, dots, horizontal cell processes, asterisks, rod bipolar cell dendrites.

Fig. 1: Top: Different bipolar cells in the retina stratify at different levels of the inner plexiform layer (IPL). Bottom: Representative current recordings of type 4, type 5, and type 6a bipolar cells (for details see text). Current traces of different cell types show characteristic differences, indicating differences in ion channel expression.

[1] H. Wssle and B.B. Boycott, Physiol. Rev. 71, 447 (1991). [2] R.H. Masland, Curr. Opinion Neurobiol. 11, 431 (2001). [3] H. Wssle, C. Puller, F. Mller and S. Haverkamp, J. Neurosci. 29, 106 (2009).

An alternative pathway for rod photoreceptor signals The retina functions over a wide range of light intensities. The very sensitive rod photoreceptors are suitable for vision at low (scotopic) light levels whereas the less sensitive cones provide for daylight (photopic) and colour vision. The cones are connected to the cone bipolar cells that synapse onto the ganglion cells. For a long time it was thought that rod signals are only relayed by one type of bipolar cell to the inner retina, the rod bipolar cell. Recent evidence suggested an alternative route in which rods directly contact some types of OFF-cone bipolar cells. We performed an immunohistochemical analysis on the level of light and electron microscopy to identify the bipolar cells and ganglion cells that are involved in this alternative rod pathway of the mouse retina. We identified two types of OFF-bipolar cells, one of which was hitherto unknown, that form contacts at both cone pedicles and rod spherules. The axon terminals of these bipolar cells co-stratify with the dendrites of a large, putatively postsynaptic OFF-ganglion cell. These newly identified cell types represent the basis of a neuronal circuit in the mammalian retina that could provide for an alternative fast rod pathway [5].

[4] E. Ivanova and F. Mller, Vis. Neurosci. 23, 143 (2006). [5] A. Mataruga, E. Kremmer and F. Mller, J. Comp. Neurol. 502, 1123 (2007).

69

Maturation and Modulation of Neuronal Chloride Homeostasis

T. Gensch1, D. Gilbert2, A. Woitecki2, C. Franjic-Wrtz2, K. Funk2, S. Frings2, F. Mhrlen2
1 2

ISB-1: Cellular Biophysics Institute of Zoology, Univ. Heidelberg, D-69120 Heidelberg influx that causes hyperpolarization and inhibition of the neuron. We have studied the effect of inflammatory mediators (i.e. CGRP or substance P) on the [Cl ] as well as on the expression of NKCC1 and KCC2 in DRG neurons. To monitor changes of the Clconcentration during exposure to inflammatory mediators, we loaded DRG neurons with the Cl-sensitive fluorescent dye MQAE. The MQAE fluorescence is quenched by Cl- and is well suited to monitor time-dependent changes of the intracellular Cl level. As an experimental parameter we analyzed the fluorescence lifetime of MQAE, which is independent of the intracellular dye concentration. The recorded fluorescence lifetime was colour-coded for microcopic images such that warmer colours represent higher Clconcentrations (Fig. 1).

In most neurons of the adult CNS, the internal chloride concentration [Cl-] is low. Hence, opening of GABA- or glycine-activated ionotropic receptors mediate Cl- influx, i.e. lead to inhibition of the cell. Excitatory Cl- currents occur in neurons which accumulate Cl , i.e. in immature neurons of the CNS, in olfactory sensory neurons, and in neurons challenged by ischemia, inflammation, or neurological disorders. The balance between inhibitory and excitatory Cl- effects is determined by Cluptake and Cl extrusion pathways in the cell. + + A Na -K -2Cl co-transporter NKCC1 provides the main route for Cl uptake, whereas KCC2 couples Cl extrusion to K+ efflux. We have determined the intracellular Cl concentration [Cl ] of somatosensory neurons using twophoton fluorescence-lifetime imaging microscopy (2P-FLIM) with the Cl--sensitive dye MQAE [1] and monitored the expression of NKCC1 and KCC2. In a second approach, we studied the effects of inflammatory mediators on Cl homeostasis in dorsal root ganglion (DRG) neurons [2]. Our results show, that DRG neurons undergo a developmental transition of chloride homeostasis during the first three postnatal weeks. In contrast to CNS neurons, DRG neurons display a heterogeneous pattern of Cl concentration. Inflammatory mediators raise the intracellular Cl- concentration, most likely due to post-translational modification of NKCC1 and KCC2 and thereby change the excitability of DRG neurons. This may be the basis for increased pain perception during inflammation. Neuronal activity can be strongly influenced by Clcurrents. Whether the opening of Cl- selective ion channels leads to excitatory or inhibitory currents, is controlled by the membrane potential and by the intracellular Cl concentration. Cation-coupled Cl cotransporters control the [Cl-] in neurons. Cluptake is mainly mediated by NKCC1 [3] activity, whereas the efflux of Cl- ions is controlled by KCC2 [4]. Regulatory mechanisms that control the activity of Cl- transporters include phosphorylation as well as dimerization. Changing the balance between Cl- import and export can have profound effects on the Nernst-potential for chloride ECl and thereby determine whether the opening of Clefflux, leading to channels causes Cl depolarization and excitation of the cell, or to Cl-

Fig. 1: Monitoring intracellular Cl concentration in DRG neurons. (A) Two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intact DRGs loaded with MQAE. The colour code indicates the fluorescence lifetime () which is inversely proportional to the Cl concentration. Warm colours represent high intracellular Cl (small values). (B) Quantitative analysis of 2P-FLIM data show a highly significant increase of the inverse lifetime (LT = 1/) two hours after starting the treatment with inflammatory mediators.

The data indicate that inflammatory mediators raise intracellular [Cl-] significantly within 2 hr of treatment with inflammatory mediators, e.g. substance P and calcitonin-gene related peptide (CGRP). Notably, the 2P-FLIM measurements revealed that virtually all DRG neurons visible in the image increase their [Cl ] in response to the inflammatory stimulus. This effect coincided with

70

enhanced phosphorylation of NKCC1. Immunological staining of DRG sections with antibodies specifically binding to phosphorylated NKCC1 showed an increase of 18% and 45% of the phosphorylated form of NKCC1 after 1 and 2 hrs of inflammatory treatment. These results suggest that phosphorylation of NKCC1 in DRG neurons causes the early (< 3 hr) phase of enhanced Cl- accumulation observed by 2P-FLIM. Immunohistochemistry of NKCC1 and KCC2, the main neuronal Cl- importer and exporter, respectively, exposed an inverse regulation by the inflammatory mediators. While the NKCC1 immunosignal increased, that of KCC2 declined after 3 hours of treatment. In a second study we examined the maturation of Cl- homeostasis after birth (Fig. 2). Somatosensory neurons in the dorsal root ganglia undergo a transition of Cl- homeostasis during the first three weeks of postnatal development. This process parallels the developmental chloride switch in the CNS. However, while most CNS neurons achieve homogeneously low Cl- levels, somatosensory neurons maintain a heterogeneous pattern of Cl values. Somatosensory neurons have a high internal [Cl ] after birth (Fig. 2). However, within postnatal weeks 1 3, roughly a third of the cells maintain the high Cl- levels of the early postnatal days, whereas the majority of somatosensory neurons contain either intermediate or low levels of Cl-. This results, as displayed in Fig. 2 with 2PFLIM measurements of MQAE, in a reatehr heterogeneous distribution of Cl- levels in a DRG.

To unravel the molecular basis for the maturationdependent transition of the Cl concentration, we investigated the expression level of several Clcotransporters. Surprisingly, we did not detect changes in the transcript level during maturation in any of these genes. Therefore, it is highly unlikely that transcriptional regulation underlies the maturational change in Cl homeostasis in somatosensory neurons. Our studies demonstrate that Cl- homeostasis in somatosensory neurons develops during postnatal maturation into a state where the [Cl ] can be regulated individually in each neuron. Rather than on changes of the expression level of electroneutral cation-chloride cotransporters, this regulation may be achieved by postranslational modification of existing proteins. This notion is supported by our results on DRG neurons treated with inflammatory mediators in which Claccumulation coincides with phosphorylation of the NKCC1 transporter. Taken together, the efficiency of Cl accumulation sets the level of [Cl ] and, hence, may control the sensitivity of adult sensory neurons.

[1] D. Gilbert, C. Franjic-Wrtz, K. Funk, T. Gensch, S. Frings and F. Mhrlen, Int. J. Dev. Neurosci. 25, 479 (2007). [2] K. Funk, A. Woitecki, C. Franjic-Wrtz, T. Gensch, F. Mhrlen and S. Frings, Mol. Pain 4, 32 (2008). [3] B.B. Pond, K. Berglund, T. Kuner, G. Feng, G.J. Augustine and R.D. Schwartz-Bloom. J. Neurosci. 26, 1396 (2006). [4] V. Stein, I. Hermanns-Borgmeyer and T.H. Jentsch. J. Comp. Neurol. 468, 57 (2004).

Fig. 2: Determination of Cl in somatosensory neurons by 2P-FLIM. (a) Comparison of fluorescence intensity (left) and lifetime (right) images from the same DRG. In the 2P-FLIM image, the fluorescence lifetime, , is colourcoded as in Fig.1. (b) Calibration of 2P-FLIM signals in isolated DRG neurons with [Cl ] set to the indicated values using ionophores. The colour scale illustrates the false-colour representation of [Cl ] in the following images. (c) 2P-FLIM images illustrating [Cl ] levels in somatosensory neurons from newborn (P1-P4) and adult rd (3 week) mice. Newborn neurons show almost uniformly high [Cl ] (70 mM). During maturation, most somatosensory neurons decrease their [Cl ] to some extent, resulting in a heterogeneous mosaic of [Cl ] levels in the ganglia of mature animals.

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72

ISB-2: Molecular Biophysics

Director: Prof. Georg Bldt

One ultimate goal in biophysics is to understand the functions of proteins and macromolecular complexes as well as whole cellular processes like signal transduction pathways in their cellular environment. For most biophysical techniques it is difficult or in many cases impossible to apply physical methods to living cells. ISB-2 follows several approaches to achieve this goal:

Proteins or complexes are isolated, crystallized and high-resolution X-ray structures are determined from ground and intermediate states of their working cycles to obtain a nearly complete time-resolved image of their mechanisms. Protein unfolding and refolding as well as the interactions between proteins are studied in aqueous solution and in membranes. Cell-free transcription and translation systems in combination with fluorescence single molecule spectroscopy allow us to accompany protein synthesis and folding at the ribosomal machinery.

73

FKBP42, an immunophilin modulating ABC transporter activity

O. H. Weiergrber1 , R. Batra-Safferling1 , J. Granzin1
1

Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2)

FKBP42 is a type-II membrane protein which plays an important role in the regulation of polar auxin transport in higher plants. These effects are mediated by direct interaction with auxin carriers belonging to the ABC transporter superfamily. We have determined the X-ray structure of the soluble portion (aa 1-339) of FKBP42. The N-terminal domain displays an FKBP-type fold belonging to the -class. The C-terminal segment, on the other hand, represents a helical bundle comprising three TPR motifs. Based on sequence conservation, we predict that the interaction of FKBP42 with the heat shock protein HSP90 will occur in a similar way as found for other immunophilins. Finally, we have built a model for the complex of FKBP42 with the Cterminal nucleotide binding domain of the auxin transporter PGP1. We propose that FKBP42 may enhance auxin transporter activity by stabilizing a conformation of the nucleotide binding domain which is competent for ATP loading and/or hydrolysis. The FK506 binding proteins (FKBPs) represent a ubiquitous protein family named after the role of several members as primary targets of FK506-type immunosuppressants in animal and human cells. Based on this activity, the FKBPs, together with the family of cyclophilins, have also been termed "immunophilins". Another feature shared by many FKBPs is the ability to act as peptidylprolyl cis-trans isomerases (PPIases), which implicates these proteins in peptide folding and chaperoning processes. Multi-domain FKBPs are characterized by additional protein modules, typically a tetratricopeptide repeat (TPR) domain, connected to one or more FKBP domains. Members of the FKBP family have also been identied in plants. FKBP42 from Arabidopsis thaliana, also termed TWISTED DWARF1 (TWD1) due to the reduced height and disoriented growth of null mutants, is a type-II membrane protein comprising 365 amino acids. In addition to a single FKBP-type domain, it contains a tripartite TPR motif and a hydrophobic C-terminal membrane anchor [1]. Like many other multi-domain immunophilins, the protein interacts with HSP90, which has been implicated in plant development and response to environmental stress. Intriguingly, FKBP42 is devoid
Ribbon representation of the FKBP42 structure. Mean B factors are indicated by a colour gradient from white (B < 35 2 ) to red (B > 90 2 ). Side chains establishing inter-domain contacts are indicated.
FIG. 1:

of PPIase activity and does not display measurable afnity for FK506. The FKBP domain of FKBP42 has been demonstrated to physically interact with plasma membrane-localized ABC transporters PGP1 and PGP19, whereas the TPR domain appears to be responsible for functional association with vacuolar transporters MRP1 and MRP2. Phytohormones of the auxin family represent essential regulators of plant growth and development. The predominant auxin, indole 3-acetic acid, is synthesized at the shoot apex and undergoes a basipetal transport which is crucial for the establishment of plant polarity. Recently, PGP1 and PGP19 have been shown to directly mediate cellular auxin efux, and this activity is regulated by FKBP42 [2]. We have crystallized fragments of FKBP42 covering the N-terminal domain (aa 1180) and the entire soluble portion (aa 1339), respectively [3]. The X-ray structure of the former construct reveals a canonical FKBP-type fold, consisting of a ve-stranded antiparallel -sheet wrapped around a short -helix.

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FIG. 2:

Model of the FKBP42-PGP1 complex, side view.

FIG. 3:

Model of the FKBP42-PGP1 complex, bottom view.

The absence of detectable PPIase activity as well as FK506 afnity could be explained by the active site being partly occluded by protein side chains [4]. The crystal structure of the larger construct is shown in Figure 1. The N-terminal part of the molecule (left) displays the expected FKBP-type fold. The C-terminal segment, on the other hand, represents a helical bundle (right), as anticipated from the early sequence-based classication as a TPR domain. The domain interface of FKBP42 is composed of a hydrophobic network surrounded by hydrogen bonds and electrostatic contacts [5]. Recent evidence has indicated that many of the protein-protein interactions in the HSP90 chaperone complex, which had originally been investigated in animal systems, are conserved in plants. These include the association with multi-domain immunophilins. Based on sequence conservation, we propose that the interaction of HSP90 family members with FKBP42 is mediated by the invariant Cterminal pentapeptide MEEVD engaging a TPR domain of the binding partner via a "two-carboxylate clamp" mechanism. FKBP42 is unique among large immunophilins in that both the FKBP and TPR domains have been shown to interact with ABC transporters in vivo. Specically, the C-terminal portions of PGP1 and PGP19 containing the second nucleotide-binding domain (NBD2) bind to the FKBP fold, whereas the equivalent domains in MRP1 and MRP2 associate with the TPR module. In order to improve our understanding of these novel protein-protein interactions, we have developed homology models for the NBD2 of PGP1 and MRP1 representing the two classes of ABC transporters considered here. The resulting models were subjected to an in silico docking procedure together with the appropriate domains of our FKBP42 structure. Figures 2 and 3 present an updated model illustrating the interaction of FKBP42 with PGP1 (unpublished data). To visualize the overall architecture of the complexes and the orientation of the components with respect to the membrane, the NBD2 model (blue) has been incorporated into the crystal

structure of Sav1866 from S. aureus. The crystal structure of FKBP42 described here represents the rst three-dimensional structure of a multi-domain immunophilin from plants. While the overall architecture of the two domains matches the known characteristics of the FKBP and TPR folds, respectively, their arrangement is unique and may have important functional implications. Experimental evidence indicates that regulation of ABC transporters by members of the FKBP family may not be restricted to plant cells. Our models featuring the complexes of FKBP42 with PGP1 and MRP1 may serve as working hypotheses stimulating further investigation of these novel protein-protein interactions. We are currently striving for experimental validation of our models by obtaining X-ray structures of FKBP42-ABC transporter complexes. To this end, we are establishing expression and purication of the C-terminal nucleotide-binding domains of PGP1 and PGP19, either alone or as fusion constructs with their respective N-terminal counterparts. In the context of our current efforts towards improving crystallization of membrane proteins, investigation of a fulllength ABC transporter, with or without an associated FKBP42, may ultimately be feasible.

[1] T. Kamphausen, J. Fanghnel, D. Neumann, B. Schulz and J. U. Rahfeld, Plant J. 32, 263276 (2002). [2] R. Bouchard, A. Bailly, J. J. Blakeslee, S. C. Oehring, V. Vincenzetti, O. R. Lee, I. Paponov, K. Palme, S. Mancuso, A. S. Murphy, B. Schulz and M. Geisler, J. Biol. Chem. 281, 3060330612 (2006). [3] A. Eckhoff, J. Granzin, T. Kamphausen, G. Bldt, B. Schulz and O. H. Weiergrber, Acta Cryst. F61, 363 365 (2005). [4] O. H. Weiergrber, A. Eckhoff and J. Granzin, FEBS Lett. 580, 251255 (2006). [5] J. Granzin, A. Eckhoff and O. H. Weiergrber, J. Mol. Biol. 364, 799809 (2006).

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Visual signal transduction studied by a variety of biophysical methods

R. Schlesinger1, B. Knig2, R. Batra-Safferling1, G. Bldt1, G. Dasara Raju1, J.Granzin1
1 2

Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2) Institute of Structural Biology and Biophysics: Structural Biochemistry (ISB-3)

Arrestins play a crucial role in regulation of signal transduction of G protein-coupled receptors (GPCRs) [1]. GPCRs convey a large variety of extracellular stimuli including light, odorants and neurotransmitters to the interior of cells via activation of their cognate G proteins. GPCR signaling is shut off by a conserved two step process, i.e. phosphorylation of the GPCR followed by tight binding of arrestin. Four types of arrestin can be distinguished in vertebrates: rod arrestin (arrestin-1) and cone arrestin (arrestin-4) are restricted to the corresponding compartments of the retina while non-visual -arrestin-1 (arrestin-2) and -arrestin-2 (arrestin-3) are widely distributed in various tissues. Arrestins regulate a large number of different GPCRs and serve additional functions beyond desensitization of GPCR signaling [1]. In 1998 we published the first 3-dimensional X-ray structure of arrestin-1 from bovine rod outer segments in the receptor-free form [2]. The overall fold of the basal state of arrestin is conserved across the various arrestins (see above). However, the structure of arrestin in complex with the activated and phosphorylated GPCR remains elusive. Until now it is unclear how arrestin interacts with rhodopsin. We have used several complementary methods to address local aspects of the arrestinrhodopsin interaction. Our light scattering experiments suggested that two rhodopsin molecules bind to the two domeshaped structural motifs of arrestin (Figure 1) [3]. To investigate the interaction sites of arrestin with rhodopsin various surface regions of recombinant arrestin were sterically blocked by different numbers of fluorophores. By simultaneously modifying both domains with one Alexa 633 moeity the binding capacity was reduced. The presence of two Alexa 633 molecules in each domain prevented binding of rhodopsin to arrestin. This observation indicates that both concave sites participate in binding. These findings are not consistent with the current working model of arrestin binding to rhodopsin (see literature), which requires the accessibility to different regions that

are responsible for the binding and recognition process of P-Rho*. In this case, it is expected that the presence of one dye molecule per arrestin in the concave regions as well as in the area of the helix should be sufficient to exert a clear negative influence on binding efficiency to P-Rho*. This would also be expected when considering earlier investigations which implicate a large number of residues in these regions.

FIG. 1: A possible model of arrestin (residues 6-388, J. Granzin unpublished ) and rhodopsin (Protein Data Bank ID: 1U19) interaction. The docking has been carried out manually without energy minimization and the distance between the receptor monomers is adjusted based on the work of Fotiadis et al. [5].

High resolution liquid state NMR can determine the structure and orientation of peptides in the rhodopsin-bound state (paper submitted, 2009).

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FIG. 2a: Crystal structure of uncomplexed arrestin shown as a ribbon representation. Residues 67 to 77 in the unstructured loop connecting -strands V and VI are colored in yellow, the N- and C-terminal domains of arrestin are depicted in dark blue and green, respectively.

FIG. 2b: Arrestin loop V-VI adopts an -helical structure (boxed) upon binding Meta II. Loop residues 67-77 have been replaced by the NMR structure of Meta II-bound peptide Arr(67-77) followed by energy minimization.

These binding studies indicate that Arr(67-77) competes with arrestin for its binding site on Meta II-rhodopsin. We speculate that the largely helical conformation of the Meta II-bound peptide reflects the conformation of the corresponding finger loop region in the arrestin-P-Rho* complex (Figure 2a, b). Receptor binding of Arr(67-77) strictly requires rhodopsin activation but is rather insensitive to receptor phosphorylation. These observations support the sequential multi-site binding model of receptor-arrestin interaction proposed by Gurevich and Gurevich [4] and suggest a crucial role of arrerstin loop V-VI in the recognition of rhodopsin activation.

[1] K. Xiao, D.B. McClatchy, A.K. Shukla, Y. Zhao, M. Chen, S.K. Shenoy, J.R. Yates, and R.J. Lefkowitz, Proc. Natl. Acad. Sci. USA 104, 12011-12016 (2007). [2] J. Granzin, U. Wilden, H.-W. Choe, J. Labahn, B. Krafft, G. Bldt, Nature 391, 918-921 (1998). [3] D. Skegro, A. Pulvermller, B. Krafft, J. Granzin, K.P. Hofmann, G. Bldt, R. Schlesinger, Photochem. Photobiol. 83, 385-393 (2007). [4] V.V. Gurevich and E.V. Gurevich, Pharmacol.Sci. 25, 105-111 (2004). Trends

[5] D. Fotiadis, Y. Liang, S. Filipek, D. A.

We have started time-resolved fluorescence anisotropy measurements which will give further evidence for the mode of interaction between the two molecules. In addition we will perform timeresolved fluorescence imaging experiments to visualize the interaction of arrestin with activated rhodopsin (in collaboration with U. Alexiev, FU Berlin). Furthermore crystallization of the complex (arrestin-rhodopsin) is in preparation.

Saperstein, A. Engel and K. Palczewski, Nature 421, 127-128 (2003).

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Signal transduction by sensory rhodopsin II-transducer complex

V. Gordeliy1, J. Labahn1, M. Engelhard2, G. Bldt1
1 2

Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2) Max-Planck-Institute for Molecular Phisiology

Microbial rhodopsins, which constitute a family of seven-helix retinal membrane proteins, are distributed throughout the Bacteria, Archaea and Eukaryota. The microbial phototaxis receptor sensory rhodopsin II (NpSRII) mediates the photophobic response of the haloarchaeon Natronomonas pharaonis by modulating the swimming behavior of the bacterium. After excitation by blue-green light NpSRII triggers, by means of a tightly bound transducer protein (NpHtrII), a signal transduction chain. Two molecules of NpSRII and two molecules of NpHtrII form a 2:2 complex in membranes. We have obtained X-ray structures of the ground state and photocycle intermediates K and late M (M2) explaining the evolution of the signal in the receptor after retinal isomerization and the transfer of the signal to the transducer in the complex. Membrane receptors play key roles in different cell functions. Unfortunately, there is no molecular picture of the signal transduction mechanism for any receptor. The same was true for the case of the NpSRII/NpHtrlI complex. Homologs of sensory pathway in which the complex is involved occur in all the three kingdoms of life, most notably in enteric bacteria in which the chemotaxis has been extensively studied. Recent structural and functional studies on the sensory rhodopsinII/transducer complex have yielded new insights into the mechanisms of signal transfer across the membrane. Electron paramagnetic resonance data led to the conclusion that the complex functions as a dimer [1]. However, the atomic resolution structure of the complex as well as molecular events leading to signal transduction were still missing. Our goal was to obtain such information via elucidation of high resolution structure of the complex and its intermediates. The results provided a first atomic model for signal transfer within the membrane part of the receptor. The established mechanism might also be relevant for eubacterial chemoreceptor signaling. Crystallization of the receptortransducer complex, a member of the two-component signaling cascade, has been achieved successfully using a shortened transducer comprising the two transmembrane helices (TM1 and TM2). The data showing a signal transfer from receptor to transducer indicate that HtrII-114 forms a

functional complex with its cognate receptor SRII. Thin orange crystals of SRII in complex with HtrII114 grown in lipidic cubic phase displayed an orthorhombic shape of about 140 m in size and diffracted to 1.8 . The asymmetric unit contains one complex. The expected dimer of the complex is formed by a crystallographic two-fold rotation axis, which is located in the middle of four transmembrane helices: TM1, TM2, TM1 , TM2 (where a prime indicates the right-hand complex; Fig. 1). The transmembrane helices F and G of the receptor are in contact with the helices of the transducer [2, 3]. Obviously, the binding of the transducer to helices F and G hardly interferes with the side-chain arrangement of the receptor. A notable exception is found for Tyr 199. The aromatic plane of Tyr 199 has turned in the complex by about 90 and is now pointing into the direction of TM2 where its phenolic group forms a hydrogen bond to N(2)Asn 74 (2.8 ).

FIG.1: Ribbon diagram of the top view from the cytoplasmic side. -Helices are in red for the receptor and green for the transducer; -strands are in blue and coils in grey. The labels of the symmetry related complex are marked by a prime. The crystallographic symmetry axis is located between TM1TM2 and TM1TM2 .

The FG loop region fixes the transducer by several contacts as well as by three hydrogen bonds between Thr 189 (SRII), Glu 43 (TM1) and Ser 62 (TM2). A second anchor point is observed in the middle of the membrane where, as

78

mentioned above, the phenolic hydroxyl of Tyr 199 bridges to Asn 74. The crystals of the complex were illuminated to freeze-trap the K and M intermediates. In the K state structural changes with respect to the ground state are observed within a sphere of 9 diameter around the central water cluster on the extracellular side of the retinal [4]. The isomerization of the retinal bound to Lys 205 gives rise to a displacement of its C and C atoms by 1.1 . This shift reduces the distance between Lys 205-C and the ground state position of water molecule 1 (W1-O) from 3.3 to 2.5 . Therefore the pentagonal structure of hydrogen bonds including water molecules W1 to W3 and the oxygen atoms of the aspartic residues Asp 75 and Asp 201 found for the ground state can no longer exist in the K state. W1 is driven away from its original location [2].

FIG. 3: Schematic picture of helical displacements viewed from the cytoplasmic surface. The ground-state complex is shown in red and the late M-state complex in yellow

FIG. 2: Changes in the structure of helices C and G of ground state (red) and late M state (yellow) including water molecules as red and yellow balls.

Thus, light-activated signal transfer from receptor to transducer originates in the isomerization of retinal, which induces local changes in the hydrogen-bonding network in a region near the retinal (K state). During the transition to the signaling state (late M) these disturbances increase so that in late M fewer hydrogen bonds connect helices C and G. These alterations in the hydrogen-bond network change the pKa values of the Schiff base and Asp 75, thereby enabling proton transfer, which produces a redistribution of charges. Both causes give rise to changes in the tertiary structure of the receptor in late M. Signal transfer to the transducer takes place within the interface of receptor helices F and G and transducer helix TM2. The main effect on TM2 is a clockwise rotation of about 15 and a tilt of the helix, with the hinge at Ser 62, that amounts to 0.9 at the cytoplasmic side (Leu 77). New information is of significance not only for bacterial phototaxis and chemotaxis [5], but also for other dimeric receptors, and for the first time might lead finally to a general model of transmembrane signal transduction [6].

The charge separation between the protonated Schiff-base nitrogen and Asp 75-O increases from 4.2 to 4.9 in the K state. The hydrogen bond structure (Asp 201-O W2 W4 Arg 72N) is retained, although W2 and W4 shift towards the retinal by about 1.5 in the K state. The distance from Asp 201-O to the Schiff-base nitrogen is reduced from 4.9 to 2.7 as a result of the movement of the Schiff base. During the transition from K state to M1 state the proton of the Schiff base translocates to Asp 75. In the reaction path from the ground state through K to late M the temperature factor of Asp 75-O2 varies from 18 2 (BG) through 31 2 (BK) to 22 2 (BM) and the distance between Asp 75-O2 and the Schiff-base nitrogen relaxes from 4.9 in the K state to 4.3 in late M, when proton transfer has occurred. In late M state a further hydrogen bond connecting helices C and G in the K state through water molecules W2 and W4 has vanished. Consequently these helices now have more freedom to move. Difference densities clearly show changes in the position of the main chain that are not present in the K state (for example Arg 72) (Fig 2).

[1]

Hoff W.D., Jung K.-H. and Spudich J.L. (1997) Annu. Rev.Biophys. Biomol. Struct. 26,223-258 Gordeliy V. I., Labahn J., Engelhard M., Bldt G. et al. Nature 419, 484-487 (2002). Royant A., Nollert P., Edman K., Neutze R., Landau E.M., Pebay-Peyroula E. and Navarro J (2001) Proc. Natl. Acad. Sci.USA 98,10131-10136. Moukhametzyanov R., Klare J., Efremov R., Engelhard M., Bldt G., Gordeliy V. et al. Nature 440, 115-119 (2006) Spudich J. (2002) Nature Str. Biology 9, 797-799. Newman R. (2001) Cur. Opinion in Chem. Biol. 5, 723-724.

[2]

[3]

[4]

[5] [6]

79

Vapour diffusion for in-mesophase high throughput crystallization

J. Kubicek1, G. Bldt2, F. Schfer1, J. Labahn2
1 2

Qiagen Research&Development, Hilden Institute of structural Biology and Biophysics: Molecular Biophysics (ISB-2)

The presented method to crystallize membrane proteins combines the advantages of the mesophase crystallization method and the classical vapour diffusion crystallization method. It allows fast screening of crystallization conditions employing automated liquid handlers suited for the 96 well crystallization format. Experiments with bacteriorhodopsin proof that by this approach crystals of high quality can be obtained. For less than hundred membrane proteins their 3dimensional structure at atomic resolution had been determined though one-third of the proteins inside a cell belong to this class. This state of affairs is especially bothersome as the natural entry points to a cell are membrane proteins and their assemblies: The lacking knowledge of membrane protein structures translates directly into lacking knowledge of biomedical targets and mechanisms. The small number of known membrane protein structures can be directly traced back to problems in obtaining membrane protein crystals for structural investigations. The methods developed for crystallization of soluble protein are inefficient for membrane proteins. Landau and Rosenbusch [1] used lipidic mesophases to accommodate the specific needs of membrane proteins in a way compatible with crystallization: The lipidic component monoolein self-organizes with water into mesophases [2] (Fig.1). The cubic phase Pn3m consists of a bi-continuous bilayer that separates two channel systems of aqueous phase. The bilayer is locally 2-dimensional like a cell membrane and therefore allows the incorporation of membrane proteins. But it extends continuously through space and supports therefore diffusion of the protein in three dimensions and crystallization upon dehydration.

The kinetics of the self-organisation of monoolein can be observed by incubating solid monoolein with water. Within an hour ithe formation of cubic phase is observed (Fig.2). Dehydration of the mesophase can be achieved by lowering the level of humidity. This can be achieved by adding solid salt [1].

0 min

1 min

3 min

7 min

25 min

FIG.2: Swelling experiment with 0.2 mg monoolein and 350 nl H2O at 22C. The solid, intransparent monoolein phase transforms within 25 minutes almost completely to the optically isotropic cubic phase, whereas the birefringent lamellar phase disappears.

The in-mesophase crystallization [1] unfortunately requires cumbersome manual work like weighting mg-quantities for every single crystallization experiment. This is detrimental to high-throughput screening procedures using automated liquid handling systems as employed successfully for the crystallization of soluble proteins. In vapour diffusion type crystallization experiments typically a protein solution and a solution containing a precipitating agent are mixed. A small droplet of this mixture will generally be not in equilibrium with undiluted reservoir solution. Generally the vapour pressure of water above these two phases will be different: Water will transfer via the vapour phase into the reservoir solution until the two condensed phases are in equilibrium, typically when the concentration of the precipitating agent in the droplet is approximately the same as in the reservoir (Fig.3, left: broken arrow). This end point of the experiment is to be refined experimentally to reach a droplet composition sufficiently supersaturated to force nucleation and subsequent crystallization of the protein.. In vapour diffusion experiments with monoolein the dehydration can be experimentally realized by enclosing the wetted monoolein together with a reservoir solution which takes up water from the gas phase that separates the two condensed phases (Fig.3).

FIG. 1: Isotherm from monooolein/water phase diagram. The formation of the optically isotropic cubic phases or birefringent lamellar phase can be observed with a polarization-microscope.

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Crystals of bacteriorhodopsin grown in mesophase using vapour diffusion reach a size of 0.25 mm * 0.1 mm and diffract to 1.14 (Fig.5), that is considerably further than the best results obtained so far 1.43 [3] with other crystallisation methods.

FIG. 3: Hanging drop vapour diffusion experiment with monoolein: The incubation of monoolein (dark grey) with an aqueous droplet mixed from membrane protein (green) and reservoir (red) solution leads to uptake (arrows) of water and membrane protein and subsequent phase transformation within hours. Over days the formation of optically isotropic cubic phase (light grey, middle) is observed. The loss of water from the droplet towards the reservoir (blue arrow) enhances the incorporation of protein into the cubic phase. Over weeks to month (right) the cubic phase reverts back to lamellar phase due to dehydration by vapour diffusion.

1.5

When solid monoolein is brought into contact with an aqueous protein droplet it will incorporate protein and take up water. After the fast take-up of water by monoolein the slower loss of water in the droplet by vapour diffusion will further increase the concentration of protein in the aqueous phase (Fig.3, middle). Therefore an increasing amount of the protein will be incorporated into the mesophase according to the Nernst partition law:

K = Caqueous/Xmesophase
C,X: concentration, respectively mol-fraction of protein in the respective phases

FIG.5: Diffraction experiment with bacteriorhodopsin crystal from in mesophase vapour diffusion. Measured at ERSF, Grenoble, ID14-1 at a wavelength of 0.8 . Inset shows observed resolution limit at 1.14

Lately (Fig.3, right) the protein containing cubic phase will be dehydrated by the reservoir solution and form lamellar phase (Fig.2: right to left). Nucleation of protein crystallization is thought to be brought-by by the local transformation of cubic phase to lamellar phase. Experiments with the light-driven bacterial proton pump bacteriorhodopsin from Halobacterium salinarum show that crystallisation occurs upon transformation of the transparent, isotropic, cubic phase to lamellar phase (Fig.4).

The main parameters of this crystallisation approach [4] that are to be refined experimentally for each type of precipitating agent and for each protein are: The ratio of protein and monoolein mass The mixing ratio of volumes in the initial drop The total initial drop size
[1] E.M. Landau, J.P. Rosenbusch Proc. Natl. Acad. Sci. USA, 93, 14532 (1996) [2] H. Qiu, M. Caffrey Biomaterials, 21, 223 (2000) [3] B. Schobert, J. Cupp-Vickery, V. Hornak, S. Smith J. Lanyi J.Mol.Biol. 321, 715 (2002) [4] J. Kubicek, J. Labahn, F. Schfer, G. Bldt Patent pending (2009)

FIG.4: Crystallization experiment with Bacteriorhodopsin in monoolein mesophase employing vapour diffusion.

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In vitro co-translational folding

A. Katranidis1,4, M. Geritts2, K. Nierhaus3, T. Choli-Papadopoulou4, R. Schlesinger1, J. Fitter1, G. Bldt1
1 2

Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2) RiNA GmbH, Berlin 3 Max-Planck-Institute for Molecular Genetics, Berlin 4 Aristotle University of Thessaloniki, Greece

We demonstrate here by single molecule experiments that a green fluorescence protein (GFP) is produced with a characteristic time of five minutes. The fastest GFP molecules appeared already within one minute. Processes precedent to chromophore formation, such as polypeptide synthesis and protein folding, are fast and last not longer than one minute. In our approach a two color single molecule sensitive fluorescence wide-field microscope is employed in order to visualize surface tethered labeled ribosomes and de novo synthesized GFP molecules in real time. Fluorescence of cotranslational folded proteins was observed from mature fluorescent GFP molecules which carry 31 additional amino acids at the C terminus remaining linked to the ribosome. Thus it was possible to co-localize fluorescence from labeled ribosomes and from GFP molecules. Numerous studies showed that protein folding and maturation can differ substantially between de novo synthesized proteins and in vitro refolded proteins [1]. In classical folding studies formerly folded proteins need to be transferred into an unfolded state before the (re-)folding process can be studied. It has been demonstrated in several cases that protein folding takes place already during the elongation of the nascent chain (co-translational folding). Significant differences have been observed between both types of folding with respect to folding rates, to the appearance of folding intermediates, and to the yields. Therefore, one major goal is to understand how polypeptide chain elongation and folding are coupled. In particular single-molecule studies can yield valuable information about these rather asynchronous processes. In this study we observed green fluorescent proteins (GFP) at a single molecule level after de novo synthesis and folding [2]. Formation of the fluorescent chromophore is a rather slow posttranslational autocatalytic process and the maturation kinetics as well as the folding efficiency between GFP wild type and several mutants differ significantly [3]. We have chosen the GFP Emerald (GFPem) mutant which is characterized by a high folding efficiency and by fast folding and maturation kinetics. GFP synthesis at surface-immobilized fluorescently labeled ribosomes was accomplished by using a fractionated cell-free transcriptiontranslation E. coli system (Figure 1). The sequence of GFPem was elongated by a sequence of 31

amino acids at the C-terminus (spanning the full tunnel length) in order to ensure proper folding of the full length protein outside the tunnel. Suppression of protein release after synthesis keeps the synthesized GFP bound to the ribosome and allows to image GFP fluorescence for extended observation times.

FIG. 1: Schematic view of surface-tethered ribosomes (only the 50 S subunit is shown). The aminofunctionalized cover slide is coated with a layer of PEG which is biotinylated at low concentration. By the use of a streptavidin-biotin binding assay fluorescently labeled ribosomes were linked to the surface via biotinylated ribosomal protein L4 (displayed molecules are not on scale). Cell-free synthesized GFPem becomes mature while linked to the ribosome.

For imaging fluorescently labeled ribosomes (with ATTO 655) and emerging GFP molecules we employed a dual color fluorescence wide-field microscope (Figure 2). Our images indicate that approximately 10 15% of all visible ribosomes produce a bound mature and fluorescent GFP. In a next series of measurements we monitored the appearance of individual synthesized GFP molecules as a function of time (Figure 3). For this purpose surface-immobilized ribosomes were incubated with a reaction buffer within a closed

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imaging chamber and after a dead time of 40 seconds a sequence of images was taken every 15 seconds.

Sample Ar+ Laser 488 nm Microscope objective

Beam splitter

chromophore formation time requires at least 5-10 minutes for de novo synthesized GFP molecules, we have to assume that the characteristic time constant obtained from our data (5.3 minutes) is related to the chromophore formation. Therefore we conclude that polypeptide synthesis and protein folding together must be faster than one minute.

Dye Laser ~ 640 nm

Dichroic Mirror AOTF DM

Wedged Mirror Tube Lens Emission Filter

CCD Camera

FIG. 2: Scheme of the two-color wide field setup used to image fluorescently labeled ribosomes and emerging GFP molecules at the same time with single molecule sensitivity.

To our surprise GFPem fluorescence shows up rather fast with a significant fraction within five minutes after initiating polypeptide synthesis. According to the rather limited photostability of GFPem we observe in most cases photo-bleaching after few exposures and in some cases also photoblinking. The time course of emerging fluorescent GFPem molecules is satisfactorily fitted by a single exponential. The corresponding characteristic time constant for the observed process is 5.3 minutes, which is one of the fastest maturation times for a GFP mutant observed so far. Typical maturation times of other GFP mutants of the S65T type range from 15 to 45 minutes, whereas wild type GFP shows even longer maturation times in the order of 2 hours. An interpretation of our result has to consider at least three consecutive sub-processes, namely polypeptide synthesis, protein folding, and chromophore formation, as part of the whole biosynthesis. However, in our study GFP molecules become detectable only after chomophore formation. Therefore a characteristic time obtained from the kinetic data is related to the succession of all sub-processes. (i) First, we have to account for the polypeptide elongation with a synthesis rate of about 1-5 amino acid residues per second in cell free systems, while the corresponding in vivo rate is 10-20 residues per second. For our GFP construct with 306 residues (36aa+GFPem+31aa) this would last one to five minutes for a cell free system. (ii) Folding rates of GFP are typically known from refolding studies. The corresponding times range from around 4-5 minutes for concentrated proteins in solution to a few ten seconds in single molecule studies or in chaperonin-mediated refolding. As demonstrated here protein folding seems to be fast in our approach. (iii) Since the characteristic

FIG. 3: As an example integrated peak intensities are shown as a function of time for fluorescent GFP molecules appearing at different times after the initiation of biosynthesis. Fluorescence of individual GFP molecules can only be detected for a few consecutive exposures before photo-bleaching occurs.

High rates of folding and maturation are assumed to play a crucial role to reduce unwanted side reactions, such like misfolding and aggregation, and thereby improve the efficiency of protein biosynthesis in the cell. Here we demonstrated that co-translational folding can be monitored on single molecule level by employing surface tethered ribosomes. Since the maturation time of the intrinsic GFP chromophore is in the order of minutes this approach does not allow to study faster events which are directly related to protein folding. Therefore, in future we aim to incorporate fluorescent dyes into the elongating nascent chain in order to monitor co-translational folding events by the use of Frster resonance energy transfer (FRET) or by fluorescence anisotropy measurements.
[1] A.H. Fedorov, T.O. Baldwin, J Biol Chem., 1997, 272, 32715-32718. [2] A. Katranidis, D. Atta, R. Schlesinger, K.H. Nierhaus, T. Choli-Papadopoulou, I. Gregor, M. Gerrits, G. Bldt and J. Fitter, Angewandte Chemie Int. Edit., 2009, 48, 1758-1761 [3] M. Zimmer, Chem. Rev., 2002, 102, 759-781.

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Crystal structure of PI3K SH3 domain in complex with an artificial peptide

R. Batra-Safferling1, J. Granzin1, S. Hoffmann2, D. Willbold2
1 2

Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2) Institute of Structural Biology and Biophysics: Structural Biochemistry (ISB-3)

Src homology 3 (SH3) domains are small protein modules (~60 residues), found in a number of intracellular signaling proteins, and are mediators of protein-protein interactions [1]. Here we investigated the crystal structures of the SH3 domain from phosphatidyloinositol 3kinase (PI3K) in the presence of an artificial 12residue proline-rich peptide PD1R (HSKRPLPPL PSL) (Table1) [2]. The crystal structure of the PI3K SH3-PD1R complex at a resolution of 1.7 reveals the type I ligand orientation. The bound peptide has an extended conformation where the central portion forms a left-handed type II polyproline (PPII) helix. The overall structure of SH3 domain shows minimal changes upon ligand binding. In addition, we also attempted crystallization studies with another artificial peptide ligand PD1Y where the anchor residue at position P-3 is a tyrosine. The crystal structure reveals that the ligand is missing but the binding site is occupied by the residues Arg18 and Trp55 from the symmetry related molecule. Considering the above mentioned crystal structures of PI3K SH3 and the published reports, we provide a comparative analysis of protein-ligand interactions that has helped us identify residues which play an important role in defining target specificity. The role of SH3 domain is to mediate proteinprotein interaction via binding to proline-rich motifs in the target proteins enabling the formation of multimeric signaling complexes. SH3 domains recognize unique proline-rich peptides bearing the core sequence PxxP (where P = proline, x = any amino acid), flanked by other residues specific to the domains. Binding of these peptides is not specific and the binding constants vary in milli- to micromolar range. In previous experiments, we identified several artificial peptide ligands from screening a phage displayed peptide library against lymphocyte specific kinase (Lck) SH3 domain [2]. The peptides screened follow a Class I (+xxPxxP) consensus sequence with an average length of 12residues (+=basic residue; P=conserved residue proline; p=proline preferred) (Figure 1). Our efforts remain to structurally investigate several different SH3 domains in the presence of artificial peptide ligands that bind in M range. The studies are aimed to first, elucidate the molecular interactions involved in binding mechanism and second, to shed light on the issue of specificity of the ligand binding to SH3 domains.

Recently, we solved the crystal structure of SH3 domain from PI3K kinase in complex with an artificial peptide PD1R at 1.7 resolution (Figure 1). The overall -barrel fold of PI3K SH3 crystal structure is similar to that observed for the SH3 domains. The protein structure consists of two orthogonal -sheets containing five antiparallel strands (4-11, 28-33, 54-60, 64-70, 74-80), two unique regular helices (34-41, 50-53), two short 310 helices and three loops (Figure 1). Ligand binding: The bound peptide adopts a lefthanded PPII helix conformation, seen for the class I orientation (Figure1). The residues with their sidechains facing the protein are Arg4, Pro7 and Pro10,

FIG. 1: Crystal structure of the PI3K SH3 domain in complex with ligand peptide PD1R. PI3K SH3 domain is shown as ribbon structure where beta strands, loops and alpha helices are colored red, yellow and blue, respectively. Two 310 helices are shown within the loops in dark blue. Residues of the peptide with side chains are shown as stick model with electron density from the final 2Fo-Fc map. The contour level is at 1. The amino acid sequence of the peptide PD1R is HSKRPLPPLPSL

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Sequence

KD value (M) 40 120

PD1R PD1Y

HSKRPLPPLPSL HSKYPLPPLPSL

TABLE 1. Sequence and KD values of the artificial peptide ligands identified by Tran et al. [2].

solution structure of hematopoietic cell kinase (Hck) SH3 domain in complex with PD1Y (Figure 2B) [3]. In the later structure, lysine at position P-3 substitutes the function of anchor residue, readjusting the C-terminal residues that cause a kink in the peptide (Figure 2B). Based on our studies, we suggest that the binding mechanism involves two steps first, binding of the anchor residue and second, binding of the polyprolines.

where the residue Arg4 is at the anchor position P - 3, and residues Pro7 and Pro10 correspond to the positions P0 and P3, critical for ligand binding. The residues reside on three well-define pockets: The Pro7 sits in the pocket formed from residues Tyr14, Trp55, Pro70, and Tyr73 of the SH3 domain; Pro10 and side chain of residue Leu9 are both in a common pocket, interacting with residues Tyr12 and Tyr73 of the SH3 domain; Arg4 lies sandwiched between the residues Arg18 and Trp55 of the protein, and is surrounded by acidic residues Glu17, Asp21 and Asp68 where it forms salt bridges with Glu17 and Asp21 of the protein. In a parallel attempt, we tried to co-crystallize PI3K SH3 domain with a modified peptide PD1Y, carrying a tyrosine at anchor position P-3 instead of an arginine (Table 1). Even though the KD values are in M range, the protein crystallized as free PI3K SH3 domain and not as PI3K SH3-PD1Y complex. However, the ligand binding site is occupied by residues Arg18 and Trp55 of a symmetry related molecule where the side chains of arginine residues are sandwiched between the side chains of two tryptophan residues (Figure 2A). The Trp-Arg-Arg interactions seen are similar to those observed between the anchor residue Arg4 of the peptide PD1R and residues Arg18 and Trp55 of SH3 domain in PI3K SH3-PD1R complex. It is known from previously solved structures and from the sequence analysis that Trp55 is a highly conserved residue that contacts the ligand and is important in ligand binding. In contrast, structure based alignments show that residue Arg18 is not conserved and a great deal of structural variations are seen at this position upon ligand binding. Such non-conserved positions that participate in ligand binding are predicted to play a role in defining specificity for the ligand. Here, we show that upon substitution of residue arginine at the anchor position by tyrosine in the ligand leads to binding in solution but in the crystal structure, ligand is not present. It is plausible that the Trp-Arg-Arg stack formation (as seen in the complex structure) is entropically a favorable event. The stabilizing role of Trp-Arg-Arg --stacking has been recognized in crystal structures for several receptors. As the residue arginine is absent at the anchor position in the PD1Y ligand, the protein prefers residue Arg18 of the symmetry related protein molecule instead, allowing the crystal packing where the ligand is omitted. We compared the PI3K SH3-PD1R complex crystal structure with our previously reported NMR

FIG. 2: A. Interactions mediated by crystal lattice between the PI3K SH3 domain and the symmetry related molecule. Cell boundary is shown in gray line. B. Super-imposition of PI3K SH3 bound PD1R (yellow) with HcK SH3 PD1Y(red). Also shown is the side chain of residue Trp55 of SH3 domains.

In future studies, we would investigate the role of non-conserved residues (like Arg18 of PI3K) which not only play a role in ligand binding, but also in the selection of their respective ligands.

[1] T. Pawson, and G. D. Gish, Cell, 71, 359 (1992). [2] T. Tran, S. Hoffmann, K. Wiesehan, E. Jonas, C. Luge, A. Aladag and D. Willbold, Biochemistry, 44, 15042 (2005). [3] H. Schmidt, S. Hoffmann, T. Tran, M. Stoldt, T. Strangler, K. Wiesehan, and D. Willbold, J. Mol. Biol., 365,1517 (2006).

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Studies on multi-domain protein folding

T. Rosenkranz1, J. Fitter1
1

Institute of Structural Biology and Biophysics: Molecular Biophysics (ISB-2)

Most of fundamental studies on protein folding have been performed with small globular proteins consisting of a single domain. In vitro many of these proteins are well characterized by a reversible two state folding scheme. However, the majority of proteins in the cell belong to the class of larger multi-domain proteins which often unfold irreversible under in vitro conditions. This makes folding studies difficult or even impossible. A promising approach in this respect is given by single molecule techniques which experienced tremendous methodological advances in recent years. In particular fluorescence based methods can contribute significantly to proceed in understanding fundamental principles of multidomain protein folding. In prokaryotic and even more in eukaryotic cells the predominant fraction of the whole proteome belongs to the class of multi-domain proteins. However, because of methodical reasons our existing knowledge about mechanisms and principles of protein folding results mainly from studies on smaller single-domain proteins. In particular competing side reactions such as misfolding and aggregation of non-native states makes folding studies on larger multi- domain proteins often difficult or even impossible under in vitro conditions. Therefore, working at low protein concentrations and employing single molecules studies are well suited to extend our knowledge on multi-domain protein folding. Extremely low protein concentrations (~ nM), as used for example in fluorescence correlation spectroscopy (FCS) studies, can lower or even circumvent aggregation of unfolded states. In principle, this can make refolding studies feasible, which would be hampered by aggregation at higher protein concentrations [1,2] (Fig. 1). In addition, the application of single molecule techniques has an enormous potential for studying the intrinsically heterogeneous process of protein folding, not only for multi-domain proteins. For protein folding studies on single molecule level mainly FCS and energy transfer techniques (FRET: Frster resonance energy transfer and PET: photoinduced energy transfer) have been employed. FCS is an easily applicable technique which needs only one fluorescent dye to be attached to the protein of interest. The diffusion time of the fluorescently labeled protein through the confocal volume of a tightly focused laser beam allows to determine the hydrodynamic radius (Rh) of the protein. Typically,

the native state is compact and exhibits a smaller hydrodynamic radius as compared to the more expanded unfolded state. For -amylase at least a partial refolding was observed only at extreme low proteins concentrations (~nM) as used in FCS, while at higher protein concentrations (~M) refolding was hindered due to aggregation (Fig.1B).

(A)

1,00 fraction unfolded (B) 0,75 0,50 0,25 0,00 0 1 2 GndHCl [M] BLA_native BLA_unfol BLA_refol 3 BLA_unfolding BLA_refolding

(C) norm G() 0,8

0,4

0,0 0,01

0,1

1 time [ms]

10

100

FIG. 1: (A) Schematic representation of the Bac. Licheniformis -amylase (BLA) structure. The color code (green, orange, cyan) represents the domain structure. (B) Transitions as observed under unfolding and refolding conditions measured with CD spectroscopy at a protein concentration of about 3 M. As shown here, proper refolding is not taking place. (C) Autocorrelation curves as measured with FCS for BLA in the native state, in the unfolded stated, and in the refolded state. Here at low protein concentrations (~ 1nM) we observe at least a partial refolding (red line moves back to that for the native state).

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Another approach to avoid protein aggregation, in particular of the unfolded state, is to encapsulate single molecules in surface-tethered nanocontainers (Fig. 2A). By this kind of encapsulation individual proteins are separated from each other and represent an ideal probe for single molecule studies.

in solution. In order to immobilize individual protein molecules, we encapsulate them in polymeric vesicles made of amphiphilic triblock-copolymers and tether the vesicles to a cover slide surface. A major goal of this study is to investigate polymeric vesicles with respect to their suitability for protein folding studies. The fact that polymeric vesicles possess an extreme stability with respect to various chemical conditions is supported by our observation that harsh unfolding conditions do not perturb the structural integrity of the vesicles. Moreover, polymerosomes prove to be permeable to GdnHCl and thereby ideally suited for unfolding and refolding studies with encapsulated proteins. We demonstrate this with encapsulated phosphoglycerate kinase (PGK), which was fluorescently labeled with Atto655, a dye that exhibits pronounced photoinduced electron transfer (Fig. 2A). In the case of PGK we were able to make use of PET due to the fact that Atto655 attached at a defined position within the protein structure is efficiently quenched by a tryptophan residue located in close proximity to the dye (low emission intensity). Upon unfolding, a structural expansion takes place and the average distance between the dye and the quencher is increased which results in a lower quenching efficiency (high emission intensity, Fig. 2C). We demonstrated here that proteins encapsulated in polymeric vesicles offer the possibility to study individual proteins for extended time periods. The encapsulation procedure provides a native-like environment for water soluble proteins without detectable hindrance of rotational reorientations. The high structural stability and considerable longevity of the polymerosomes qualifies them to be an ideal tool for single molecule studies. The permeability of triblock-copolymer membranes for GdnHCl and their resistance against structural disintegration at high denaturant concentrations ensure ideal conditions for their use in protein folding studies. At thermodynamic midtransition points (e.g., GdnHCl1/2, T1/2, pH1/2) in particular FRET based approaches provide structural details during folding and unfolding transitions of the proteins. At these conditions the folded and unfolded states have life times in the order of seconds and multiple successive unfolding/ refolding transitions can be observed with FRET for single encapsulated proteins. A major goal for future studies will be the focus on proteins that exhibit unfolding/folding transitions slow enough that trajectories of these transitions can be followed.

Fig. 2: (A) Scheme of an individual in a polymersome encapsulated protein labeled with a fluorescent dye. (B) Wide field fluorescence image of surface tethered polymerosomes containing Atto655 labeled PGK. (C) A typical time-course of the measured emission intensity as obtained from the integration of an individual spot. The corresponding images (see B) were measured every 30 seconds with polymerosomes bound to cover-slides which were built-in a closed imaging chamber suitable for in-situ buffer exchange. The arrows indicate buffer exchange from native to unfolding conditions or vice versa.

[1]

K.H. Strucksberg, T. Rosenkranz, J. Fitter, BBA, 2007, 1774(12), 1591-1603

[2] J. Fitter, Cell. Mol. Life Sci., 2009, 66(5), xx-xx [3] T. Rosenkranz, A. Katranidis, D. Atta, I. Gregor, J. Enderlein, M. Grzelakowski, P. Rigler, W. Meier and J. Fitter, ChemBioChem, 2009,10,702-709

Immobilizing biomolecules provides the advantage to observe them individually for extended time periods, unlike in case of freely diffusing molecules

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88

ISB-3: Structural Biochemistry

Director: Prof. Dieter Willbold

The research areas of the Structural Biochemistry group comprise the investigation of precise three-dimensional structures and dynamics of biologically and medically relevant macromolecules in order to fully understand their functions and their related cellular processes. Research is focused on proteins that are involved in diseases like AIDS, SARS and neurodegenerative disorders to study the molecular basis of these diseases and to investigate novel approaches to therapy.

Some of our target proteins are fibril forming or membrane proteins and are therefore not readily amenable to structural investigation. Our research purposes include e.g. the investigation of the structural basis of protein-protein and protein-ligand interactions to explore how viral proteins of HIV and SARS-CoV interact with host cell proteins to use and reprogram the host cell for viral replication and protection against the host immune system. Furthermore, ultra-sensitive assays potentially suitable for early and non-invasive diagnosis of neurodegenerative disorders like Alzheimers disease and prion diseases are developed. To study these phenomena several molecular biology and biophysical techniques, e.g. liquid and solid state NMR, fluorescence correlation spectroscopy as well as, in cooperation with ISB-2, X-ray crystallography are developed and applied. Moreover, computational biology approaches in the fields of structural biology are performed.

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D-peptides for diagnosis and therapy of Alzheimers disease

S. A. Funke1, T. van Groen2, I. Kadish2, L. Nagel-Steger3, K. Wiesehan1, D. Willbold1,3
1 2

ISB-3: Structural Biochemistry Dept. Cell Biology, University of Alabama at Birmingham, AL 35294, USA 3 Institute of Physical Biology, Heinrich Heine-Universitt Dsseldorf The amyloid cascade hypothesis assigns the amyloid-beta-peptide (A) a central role in the pathogenesis of Alzheimers disease (AD). We searched for peptides consisting solely of Denantiomeric amino acids (D-peptides) with strong binding to A(1-42). D-peptides are thought to be protease resistant and less immunogenic than the respective L-enantiomers. Using mirror image phage display we identified two interesting D-peptides having different properties concerning A cytotoxicity and A aggregation. Peptide D1 is being developed into a probe for in vivo imaging of amyloid plaques in the living brain, wheras peptide D3 has interesting therapeutical properties. Alzheimers disease (AD) is a progressive, neurodegenerative disorder which is characterized by loss of brain functions. More than 20 million people are affected worldwide. The histopathological hallmarks are protein deposits (senile plaques and neurofibrillary tangles) in the brain. One of the typical hallmarks of AD, senile plaques, consists mainly of extracellular Amyloid- peptide (A) deposits. A is a 4 kDa peptide of 39 to 43 amino acids derived from proteolytic cleavage of the amyloid precursor protein (APP) by two enzymes, - and -secretase. Soluble A polymerizes to form neurotoxic oligomers, which also fold to fibrils, and deposits around neurons. These deposits spread in different areas of the brain and the "amyloid-cascade-hypothesis" assigns those A plaques a central role in the pathogenesis of the disease. So far, no method for pre-symptomatic diagnosis of AD is available. To improve diagnosis and treatment evaluation, neuroimaging tools, making use of A-binding ligands visualizing amyloid plaques, are developed. A mirror image phage display approach was used in our lab to identify novel and highly specific ligands for aggregated A(1-42). Mirror image phage display allows the use of phage display to ultimately identify peptides that bind specifically to a given target and consist solely of D-amino acids. D-Amino-acid peptides are known to be less protease sensitive, more resistant to degradation in animals and less or even not at all immunogenic as compared to L-amino-acid peptides. In order to obtain the exact mirror image of a given protein (L-protein), it is necessary to synthesize a protein with the same amino acid sequence but composed exclusively of D-amino acid residues (D-protein). Such a D-protein can be used as a target for phage display selection like any other target. In the study described herein, we used the D-enantiomer of A as target and a 12-mer randomized peptide library displayed on the surface of M13 bacteriophages to be screened for D-A-binding peptides. For reasons of symmetry, the D-enantiomeric form of the selected 12-mer peptide should interact with the A peptide consisting of L-amino acids. Here, a phage displayed peptide library with more than 1 x 109 different 12mer peptides was screened for peptides with binding affinity to the mirror image of aggregated A(1-42) [1]. The resulting peptide, D1, binds very specifically to A aggregates with a binding constant in the submicromolar range. Fibrillar deposits derived from other amyloidoses are not stained. As can be seen in Fig. 1, D1 binds to A deposits in brains of AD transgenic mice (APPswe-PS9) in vivo [2]. Although D1 shows interesting properties in reducing A cell toxicity and amyloid formation assays [3], it will mainly be developed as a molecular probe for in vivo imaging of amyloid plaque load in AD patients.

Fig.1: FITC-labelled D1 binds to A plaques in the brains of transgenic APPswe-PS9 mice in vivo. The peptide was directly injected in the brain of the mice. One week after injection, the mice were sacrificed and the brains were investigated by histochemical analysis.

90

Although there is still controversial discussion if A is the causative agent in AD, inhibition of A production and aggregation are often addressed for therapy development. During a second phage display selection with monomeric or low molecular weight oligomeric D-A(1-42), we obtained peptide D3, which modulates A aggregation and reduces A cytotoxicity in vitro (Fig. 2).

processes. The number of activated astrocytes and microglial cells (measured as density of GFAP and CD11b staining) near plaques of equal size was significantly less compared to the control brains (Fig. 4), indicating that D3 changes the inflammatory properties of A.

120

** **
cell viability (%)
100

**
*

80 60 40 20 0 0 10 25

no D3 10 M D3 100 M D3 1 mM D3

A 1-42 concentration during aggregation (M)

Fig.2: PC12 cell viability in absence or presence of A and/or D3. Different concentrations of D3 were added to 10 M A samples or to samples without A as controls. The A/D3mixtures were incubated for 6 days at 37C and 1:5 diluted into PC12 cell cultures. Cell viability was measured using MTT assay. Percentages of cell viability were derived as follows: The 100 % value was obtained from cells treated neither with the respective peptide nor with A; the value of 0 % was obtained by treatment of the PC12 cells with 0.2 % Triton-X.FITC-labelled

In cooperation with Thomas van Groen (University of Alabama, Birmingham, USA) we carried out investigations on the effects of D3 on A deposits in brains of transgenic mice (APPswe/PS9). These mice develop elevated levels of A at four months of age and at the age of 5 months they show amyloid plaque pathology. An inspection of the brain sections showed a significant reduction of the A-load in the hippocampus and frontal cortex after treatment with D3 (see Fig. 3) [4].

Figure 4. Influences of D3 on inflammation in brain tissue sections of transgenic APP-PS mice. Saline (Control) or D3 was infused in the brains of the mice for four weeks. Animals were transcardially perfused at the end of the infusion period, and coronal sections were cut through the brain, one series of sections was stained with GFAP or CD11b. Representative sections showing Congo red positive plaques the dorsal cortex are shown. Please note the decrease in inflammation (activated astrocytes and microglia) surrounding Congo red-positive plaques in the D3 infused brain compared to the control brain.

In recent studies, D3 improved the cognitive performance of treated mice in the water maze assay after oral application (unpublished results). We suggest an A (1-42) modulating activity of the peptide as it clearly interacts with early intermediate assemblies of A and promotes fibrillization (unpublished results). Recents studies indicate that soluble A oligomers (also called protofibrills, prefibrillar aggregates and ADDLs) instead of fibrillar forms are the major toxic species in AD. Therefore, agents that interfere with early oligomerization processes might be valuable for therapy of AD. Therefore, D3 is a good starting point for therapy development.

Fig. 3: Influence of D3 on A load in brain tissue sections of transgenic APPswe-PS9 mice. Saline (Control), D1 as a peptide control or D3 were infused into the brains of the mice for four weeks. Animals were transcardially perfused at the end of the infusion period, and coronal sections were cut through the brain, one series of sections was stained with W0-2 (anti human amyloid-). Representative sections showing the hippocampus and dorsal cortex are shown. Please note the decrease in A staining in the D3 infused brain compared to the control brains.

[1] Wiesehan K, Buder K, Linke RP, Patt S, Stoldt M, Unger E, Schmitt B, Bucci E, Willbold D. ChemBioChem 4, 748-753 (2003). [2] Van Groen T, Kadish I, Wiesehan K, Funke SA, Willbold D. ChemMedChem 4, 276-282 (2009). [3] Wiesehan K, Sthr J, Nagel-Steger K, van Groen T, Riesner D and Willbold D Prot. Eng. Des. Sel. 21, 241-246 (2008). [4] Van Groen T, Wiesehan K, Funke SA, Kadish I, Nagel-Steger L and Willbold D ChemMedChem 3: 1848-1852 (2008). [This article was evaluated by the Faculty of 1000 Biology as a "must read"]

Detailed analysis of the inflammation (i.e., density of activated astrocytes and microglial cells) around remaining A deposits revealed that the D3 treatment significantly reduced inflammation

91

Human CD4: NMR structure and interactions of C-terminal domain

M. Wittlich1,2, B. W. Koenig1,2, S. Hoffmann1,2, D. Willbold1,2
1 2

ISB-3: Structural Biochemistry Institute of Physical Biology, Heinrich-Heine-Universitt Dsseldorf

The type 1 transmembrane glycoprotein CD4 of 58 kDa plays a key role in the adaptive immune response. CD4s ectodomain serves as main receptor of human immunodeficiency virus (HIV) and binds the HIV-1 envelope glycoprotein gp120. Physical interactions of HIV-1 accessory proteins Nef and Vpu with the cytoplasmic tail of CD4 result in downregulation of CD4 from the surface of infected cells. Our goal is the detailed characterization of the structure of the membrane-anchored Cterminal domain of CD4 in complex with Vpu and Nef, respectively. Here we present an efficient protocol for recombinant production, purification, isotope labelling, and membrane reconstitution of CD4tmcyt, containing the transmembrane and cytoplasmic domains of CD4. Specific binding of liposomereconstituted CD4tmcyt with the cytoplasmic domain of Vpu is demonstrated. The NMR structure of CD4tmcyt in membrane mimicking micelles is presented. Human CD4 consists of an extracellular region of 371 amino acids, a short transmembrane region, and a cytoplasmic domain of 40 amino acids (Fig. 1). The CD4 T-lymphocyte coreceptor belongs to the IgG-superfamily and participates in T-cell activation and signal transduction. In addition to these functions, CD4 is the major receptor for HIV infection. The virus is internalized after binding of HIV-1 gp120 to the extracellular domain of CD4. Following infection, two additional viral proteins, Vpu and Nef, interact with CD4, but bind to the cytoplasmic domain of CD4. Vpu-induced degradation of CD4 molecules in the endoplasmatic reticulum requires both proteins to be inserted into the same membrane. The CD4 sequence relevant for this activity is located between amino acids 402-420 [1]. In contrast, Nef acts at the cell surface to mediate the internalization and lysosomal degradation of CD4. Residues 407 to 418 in the cytoplasmic tail of CD4, with special emphasis on the dileucine motif at positions 413 and 414, are necessary and sufficient for down-regulation of CD4 by Nef [2-4]. Recombinant production of transmembrane proteins in E.coli is very challenging. It often results in insoluble protein aggregates, low protein yield, and numerous obstacles during protein refolding and membrane reconstitution. We produced CD4tmcyt as part of a fusion protein containing an N-terminal decahistidine tag and the

soluble protein ubiquitin (Fig. 1) [5,6]. Supplementing all buffers employed for cell lysis, purification, and enzymatic cleavage of the fusion protein with a dedicated mixture of non-ionic surfactants was crucial for increasing solubility of the protein and for the exceptionally high yield of purified CD4tmcyt. We obtained ~6 mg CD4tmcyt per liter of bacterial culture.

FIG. 1: Schematic representation of human CD4 (above) and of the recombinantly produced fusion protein (below). CD4 features a large extracellular domain (EXT), one transmembrane span (TM), and a carboxy-terminal cytoplasmic tail (CYT). The N-terminal tag of the fusion protein consists of yeast ubiquitin preceded by a dipeptide (MG), a decahistdine tag, and the 11 residue linker L1 (SSGHIDDDDKH). Linker L2 contains PreScission and thrombin recognition sequences. Protease cleavage sites are indicated by asterisks. The amino acid sequence of the 70 residue CD4tmcyt protein obtained after PreScission cleavage is shown in the lower part. The authentic CD4 residues 372 to 433 are highlighted (dark orange: TM; light orange: CYT).

CD4tmcyt contains the 60 C-terminal amino acid sequence of wild type human CD4, including five cysteine residues. We also produced the cysteinefree protein CD4mut with substitutions C394S, C397S, C420S, C422S, and C430H at similar yield using the same strategy [6]. We plan to introduce single cysteines into this mutant for attachment of ESR or fluorescence labels.

92

Both CD4tmcyt and CD4mut were successfully reconstituted into liposomes. Binding of Vpucyt (the C-terminal amino acid residues 39-81 of Vpu) to the CD4 fragments was demonstrated with a liposome centrifugation assay [5,6]. The amount of soluble Vpucyt in the supernatant and in the membrane fraction, respectively, was quantified by reversed phase HPLC (Fig. 2). Our data indicate specific binding of the recombinant CD4 domain to Vpucyt.

transmembrane helix. In contrast, incorporation of 16-doxylstearic acid into the hydrophobic core of the micelle affects only resonances in the transmembrane helix. Our next goal is incorporation of membrane proteins like CD4tmcyt into nanodiscs, a novel phospholipid-based model membrane, followed by NMR structural studies and interaction analysis.

FIG. 2: Analysis of CD4mut Vpucyt interaction in POPC membranes using centrifugation followed by reversed phase chromatography. Dispersions of POPC liposomes (10 mg POPC per sample, 1 mL each) containing varying amounts of reconstituted CD4mut (0 mg above; 0.5 mg middle; 2 mg below) were incubated with 50 M Vpucyt. Fully hydrated liposomes were separated from supernatant by centrifugation. The composition of supernatant (left column) and pellet fractions (right column) was analyzed by analytical RPC using a gradient of buffers A and B (dashed line). Position of the VpUcyt (1), CD4mut (2), and POPC (3) peaks are indicated.

FIG. 3: Schematic representation of the transmembrane and cytoplasmic helices of CD4mut in a detergent micelle. The ribbon diagram reflects the length of the two helices 1 1 derived from H H distances and secondary shift analysis. The position of the two helices with respect to the micelle was deduced from signal broadening caused by paramagnetic probes. The two cylinders surrounding the helices symbolize the space requirement of the amino acid side chains.

[1] M. Y. Chen, F. Maldarelli, M. K. Karczewski, R. L. Willey, and K. Strebel, J.Virol. 67, 3877 (1993) [2] C. Aiken, J. Konner, N. R. Landau, M. E. Lenburg, and D. Trono, Cell 76, 835 (1994)

We determined the conformation of CD4mut in membrane mimicking dodecylphosphocholine (DPC) micelles using high resolution NMR (Fig. 3) [6]. Two -helices were identified based on NOE1 1 derived H- H distances and secondary chemical shifts. They extend from M372 to V395 and from M407 to R412, in good agreement with the predicted transmembrane and cytoplasmic helices, respectively. The mutual orientation of the two helices remains undetermined due to lack of long range NOEs. The location of CD4 residues relative to the micelle was studied with paramagnetic probes: Supplementing the buffer with manganese ions (MnCl2) strongly broadens resonances in the cytoplasmic helix and in the interhelical loop, but shows little effect on resonances of the

[3] S. J. Anderson, M. Lenburg, N. R. Landau, and J. V. Garcia, J. Virol. 68, 3092 (1994) [4] A. Preusser, L. Briese, A.S. Baur, and D. Willbold, J. Virol. 75, 3960 (2001) [5] M. Wittlich, K. Wiesehan, B. W. Koenig, and D. Willbold, Protein Expr.Purif. 55, 198 (2007) [6] M. Wittlich, B. W. Koenig, S. Hoffmann, and D. Willbold, Biochim. Biophys Acta 1768, 2949 (2007)

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Mechanism of prion protein conversion and assembly

E. Birkmann1,2, J. Sthr2, T. Kaimann2, G. Panza, D. Willbold1,2, D. Riesner2
1 2

ISB-3: Structural Biochemistry Institute of Physical Biology, Heinrich-Heine-University Dsseldorf

Prion diseases are a unique group of neurodegenerative diseases because they are know as transmissible. They can occur both spontaneously and genetically caused. The infection is a conversion of host encoded prion protein (PrP) from its cellular isoform PrPC into Sc the pathological and infectious isoform PrP [1]. We investigated the conversion process by in vitro studies using recombinant PrP and natural PrPSc. The results determined with our in vitro conversion system and the derived mechanistic models are presented as a brief summary. Furthermore, well characterized intermediates and precursor states during the conversion process as well as kinetic studies of spontaneous and seeded fibrillogenesis are described.

pathway into the fibrillar pathway. Therefore, we called this state fibril precursor state and characterized it by different methods. CD spectroscopy showed a mixture of -helical and random coil secondary structure. We assume that the -helical part is identical or close to that which is also present in the fibrillar structure. Studies using analytical ultracentrifugation demonstrated the existence of a monomer-dimer equilibrium.

The mechanism of spontaneous prion protein conversion was studied systematically with recombinant (rec) PrP. Our in vitro conversion system is based on the solubilization of PrP in low concentrations (0.2% w/v) of sodium dodecyl sulfate (SDS) under otherwise physiological conditions. The conversion is induced by diluting the SDS. Most extended and systematic studies were carried out with hamster recPrP at neutral pH. Different conformations could be established. The conformations were characterized with respect to secondary structure as determined by CD spectroscopy and molecular mass as determined by fluorescence correlation spectroscopy and analytical ultracentrifugation. With these methods -helical monomers, soluble -helical dimers, soluble but -sheet rich oligomers of a minimal size of 1214 PrP molecules, and insoluble amorphous aggregates of -sheet rich structure were observed. A high activation barrier was found between the -helical dimers and the -sheet rich oligomers. In Fig. 1 the different structures are represented schematically together with their CD-spectra and the EM picture of the polymorphic aggregates. In 0.2% SDS PrP is present in an -helical and partially random coil conformation denoted with /R. Addition of 250 mM NaCl shifts PrP into a still soluble state that allows fibril assembly to proceed over several weeks. The structure of the intermediate state PrP* is decisive to switch aggregate formation from the polymorphic

FIG. 1: Molecule populations involved in the in vitro conversion of recPrP(90-231) induced by lowering SDS concentration. The molecule populations analysed within the in vitro conversion system are summarized. Exemplarily for the methods used to analyse these structures the CD-spectra are presented beneath the different intermediates. Next to the aggregated states the electron micrograph of these structures are shown. The upper part of the figure represents the conversion without added NaCl, the lower part with added NaCl, respectively. (Figure according.[3])

The studies described above indicated a critical role of PrP dimers in the conversion process consistent with research results from other groups. Consequently, we studied the structure of dimers in more detail. We selected conditions which stabilize dimers or drive the dimer-oligomer equilibrium completely to the side of dimers, respectively [4]. recPrP(90-231) in 0.06% SDS 10 mM phosphoric buffer pH 6.8 without additional NaCl is 100% dimeric. To obtain information on the conformation of the dimer, we used the covalent crosslinker EDC (1-ethyl-3-(3dimethylamino-propyl) carbodiimide), which forms

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inter- and intramolecular bonds between directly neighboured amino and carboxyl groups. The bonds were identified by tryptic digestion and subsequent mass spectrometric analysis. Intraand intermolecular cross-links between N-terminal glycine and three acidic amino acid side chains in the globular part of PrP were identified showing that the N-terminal amino acids (90124) are not as flexible as known from NMR analysis. When the cross-linked sites were used as structural constraints, molecular modelling calculations yielded a structural model for PrP dimer and its monomeric subunit including the folding of amino acids 90124 in addition to the known structure of the core domain (Fig. 2). Molecular dynamics simulation with the dimeric structure after release of the crosslink constraints indicated the domain formed by amino acids 90124 of both momomers to be intrinsically stable. The structural model (Fig. 2) is in agreement with GPI-anchoring of the dimer to the membrane. Demonstrated by arrows the two glycolipid anchors are directed to the lower side where the membrane would be present, whereas the glycosyl groups and the N-termini are directed into the water phase.

FIG. 3: Seeded fibril formation Sc RecPrP (80 ng/l) seeded with purified PrP (diamonds) forms amyloid fibrils readily compared with controls: -4 recPrP + uninfected 1.6 x 10 brain equivalents per l (x); Sc recPrP alone (triangles); purified PrP alone (dashes). Fibril formation was monitored by using the ThT Sc fluorescence assay. Electron micrographs: PrP Sc aggregates after NaPTA precipitation, recPrP + PrP after seeding assay. (Figure rearranged according [2])

To compare the in vitro conversion and the seeding efficiency in respect of differences in different species, we adopted the in vitro conversion system to bovine recPrP [5]. We could observe some differences in the structure of the fibril precursor state, but the principle conversion mechanism could be approved as described above with recPrP.

[1] Prusiner SB. (1998) Proc. Natl. Acad. Sci. USA; 95, 13363-13383. FIG. 2: Model of the prion protein dimer. Stereo presentation of the model for the PrP dimer. The structure of segment 92124 (blue) is the result of [4], the structure of segment 125228 (cyan) is taken from the NMR analysis reported in the literature. Red arrows represent the glycolipid anchor; thick blue arrows, glycosyl groups; and thin blue arrows, N-termini. (Figure according [4]) [2] Sthr J, Weinmann N, Wille H, Kaimann K, NagelSteger L, Birkmann, E, Panza G, Prusiner SB, Eigen M & Riesner D (2008) Proc. Natl. Acad. Sci. USA. 105 (7), 2409-2414. [3] Birkmann E & Riesner D. (2008) Prion, 2 (2), 67-72. [4] Kaimann T, Metzger S, Kuhlmann K, Brandt B, Birkmann E, Hltje H-D and Riesner D. (2007) J. Mol. Biol. 376, 582-596 [5] Panza G, Sthr J, Dumpitak C, Papathanassiou D, Weiss J, Riesner D, Willbold D, Birkmann E. (2008) Biochem. Biophys. Res. Commun. 373 (4), 493-497

As a model for the infection process fibril formation Sc was also studied when induced by seeds of PrP . Sc We used natural PrP purified from brain homogenate as seeds. RecPrP was applied as substrate. The fibrillization conditions were identical to those used for spontaneous fibrillization. We monitored and quantitatively described the kinetics of seeded fibril formation. Fibrillization of PrP without seeds takes up to several weeks, whereas seed-enhanced fibrillization was achieved within hours to days (Fig. 3). During the enhanced fibrillization a lag phase is followed by exponential growth and saturation.

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Solid-state NMR reveals structural differences between fibrils of wild-type and mutant -synuclein
Henrike Heise1, 2, M. Soledad Celej3, Stefan Becker3, Dietmar Riedel3, Avishay Pelah3, Ashutosh Kumar3, Thomas M. Jovin3, and Marc Baldus4
1 2

ISB-3: Structural Biochemistry Institute of Physical Biology, Heinrich-Heine-Universitt Dsseldorf 3 Max-Planck-Institute of Biophysical Chemistry, Gttingen 4 Utrecht University, The Netherlands The 140 residue protein -synuclein is able to form amyloid fibrils and as such, is the main component of protein inclusions involved in Parkinsons disease. Three point-mutations of -synuclein have been related to familial earlyonset Parkinsons disease. We have investigated the structure and dynamics of fibrils from full-length -synuclein and of its disease-related mutant A53T by highresolution solid-state NMR spectroscopy, Electron Microscopy and Atomic Force Microscopy. In both cases the C-terminus was found to be flexible and unfolded, whereas the main core region is highly rigid and rich in -sheets. Compared to fibrils from wild-type -synuclein, the well-ordered -sheet region is extended. These results demonstrate that a disease-related mutant of -synuclein differs in both aggregation kinetics and fibril structure. Parkinsons disease, the most common neurodegenerative movement disorder, is caused by the loss of dopaminergic neurons in the substantia nigra, which is accompanied by the formation of cytosolic filamentous inclusions called Lewy bodies. The major constituent of these inclusions consists of fibrillar -synuclein, a 140residue protein, which in its native conformation is largely unfolded. The central role of -synuclein aggregation in the etiology of Parkinsons disease has been further corroborated by the identification of a locus triplication of the gene encoding -synuclein as well as the three point-mutations A53T, A30P, and E46K as causes of autosomal dominant Parkinsons disease. Since their discovery, the three disease-related mutants have been the subject of intense study, as differences in aggregation behavior and morphology with respect to the wild-type form may yield valuable insights into the fundamental processes underlying amyloidogenic diseases. As in the case of wild-type -synuclein, all three mutants are natively unfolded, and significant acceleration of fibrillization has been observed for the mutants A53T and E46K. However, the overall morphology of amyloid fibrils in general depends strongly on the fibrillization conditions, including pH, temperature, salt concentration and agitation of the solution. Upon fibrillization of wild-type -synuclein at physiological temperature and pH, at least two different polymorphic forms, one consisting of straight and the other of irregularly twisted fibrils, have been obtained and characterized [1]. Studies of wild-type -synuclein by solid-state MAS NMR-spectroscopy have revealed a -sheet rich core region spanning residues from at least 38 to 94 for the two different polymorphic isoforms, whereas the C-terminus was flexible and unfolded in both cases, and the supramolecular arrangement was found to be a superpleated -sheet with parallel and in register alignment of -strands [2] (Figure 2b). In the following, we report studies on fibrils grown from the diseaserelated A53T mutant of -synuclein by solid-state NMR Spectroscopy [3].

FIG. 1: 2D C/ -synuclein fibrils temperatures of ~ Homonuclear mixing diffusion for 20 ms.

13

13

C correlation spectra of A53T recorded at effective sample 0 C (red) and -20 C (black). was achieved by proton-driven spin

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In Figure 1, two 13C/13C correlation spectra of A53T fibrils, recorded above and below the freezing point, are displayed. While the crosscorrelation signals in the high-temperature spectrum are narrower and better resolved, the low-temperature spectrum displays some additional sets of signals, especially in the region typical for non -strand secondary structures: In hydrated amyloid fibrils, different regions of a protein may exhibit diverse residual mobility, and solid-state NMR spectra based on dipolar transfer mechanisms often show only a subset of all resonances in the protein. Freezing the sample reduces the overall mobility such that flexible parts may give rise to inhomogeneously broadened signals, depending on the degree of disorder. Alternatively, flexible regions can selectively be detected by applying adequate mobility filters in the MAS NMR experiments [4].

our results, almost all resonances indicative of a -sheet conformation are in rigid protein compartments. Upon freezing the sample, resonances with random coil and -helical secondary chemical shifts appear in the spectrum, indicating that the fibrils contain regions with increased mobility and structural elements other than -sheet, while the intensities of resonances in -strands do not increase unexpectedly. For example, the low-temperature spectrum indicates the existence of at least one valine residue with a chemical shift typical for -helical conformation, which is neither part of the flexible C-terminus nor of the rigid core region, but belongs to a region of intermediate mobility. Similar to our previous results obtained on wild-type -synuclein, the C-terminus is unfolded and highly dynamic. The striking difference of the A53T compared with wildtype -synuclein is an increase in the -strand character of two leucine residues at the edges of the core region, indicating an extension of the rigid, well-ordered -sheet rich core region towards at least residue L38 and L100. The accelerated aggregation kinetics itself could result in a different pattern of molecular assembly characterized by an extended -sheet core region. However, in fibrils of both the mutant and wild-type -synuclein, the C-terminus extending from at least residue 107 is flexible, whereas the N-terminus extending from residue 22 is rigid. In contrast, in a recent study, obtained on two design mutants A56P and A30P/A56P/A76P, where proline residues were inserted into -strands in order to retard fibrillization and shift the equilibrium towards early monomers, the N-terminus was found to be flexible as well [6]. These results provide important insights into the effect of changed aggregation properties on the final fibril morphology of one disease-related mutant of -synuclein. Further studies of this nature may help to delineate the relationship between the etiology of Parkinsons disease and protein misfolding.
[1] H. Heise, W. Hoyer, S. Becker, O.C. Andronesi, D. Riedel, and M. Baldus, Proc. Natl. Acad. Sci. USA, 102, 15871-15876 (2005). [2] H. Heise, ChemBioChem, 9, 179-189 (2008). [3] H. Heise, M.S. Celej, S. Becker, D. Riedel, A. Pelah, A. Kumar, T.M. Jovin, and M. Baldus, J. Mol. Biol. 380, 444-450 (2008). [4] O.C. Andronesi, S. Becker, K. Seidel, H. Heise, H. S. Young, and M. Baldus, J. Am. Chem. Soc. 127, 12965- 2974 (2005). [5] Cornilescu, G., Delaglio, F., and Bax, A., J. Biomol. NMR 13, 289-302 (1999). [6] D.P. Karpinar, M.B.G. Balija, S. Eimer, S. Kgler, B.H. Falkenburger, G. Taschenberger, F. Opazo, H. Heise, A. Kumar, D. Riedel, L. Fichtner, A. Voigt, S. Becker, G.H. Braus, A. Herzig, M. Baldus, H. Jckle, J.B. Schulz, C. Griesinger, M. Zweckstetter. Pre-fibrillar alpha-synuclein variants with impaired beta-structure increase toxicity in Parkinson`s disease models, submitted (2009).

FIG. 2: a) Backbone angles obtained with TALOS [5] analysis of the chemical shifts. Only torsion angle pairs where at least 8 hits were found are plotted, together with the error bars. For comparison, -strands determined earlier for both wild-type forms23 are given together with the -strands of A53T. b) superpleated -sheet with in-register parallel alignment of -strands

The results of our investigations are summarized in Figure 2 a. Fibrils consist of a well-ordered rigid core region rich in -sheets, and a highly flexible and unfolded C-terminus, whereas the N-terminus lacks high mobility as of residue 22. Site-specific resonance assignments for a large part of the amino acids in the core regions were obtained from 2D homonuclear 13C/13C and heteronuclear 15N/13C correlation spectra. Intrinsic dynamics of the protein were probed by spectroscopy at different temperatures as well as by a combined mobility filter experiment specific for mobile regions of immobilized molecules. According to

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Surface-FIDA: Single particle detection as diagnostic tool

E. Birkmann1, 2, S. A. Funke1, O. Bannach2, F. Henke1, L. Wang1, D. Riesner2, D. Willbold1,2,
1 2

ISB-3: Structural Biochemistry Institute of Physical Biology, Heinrich-Heine-University Dsseldorf diseases are transmissible and AD is the most prevalent dementia. In both diseases protein aggregates occur during disease progression. Therefore, we established a highly sensitive and specific tool to detect even single protein aggregates, called surface-FIDA [1, 2, 3] (Fig. 1).

Prion diseases, Alzheimers disease, and Parkinsons disease are neurodegenerative diseases that are characterized by the formation of protein aggregates during progress of the disease. It is still not known whether these aggregates are causative for or symptoms of these diseases. Many studies show that aggregates or even oligomers of the according proteins are neurotoxic and thus may lead to neurodegeneration. To understand disease-associated or causative mechanisms in respect to protein aggregation, an ultrasensitive tool to quantify these diseaserelated aggregates is required. In this project we introduce a new diagnostic tool to count and specify even single protein aggregates. Prions are the causing agent of transmissible spongiform encephalopathies (TSEs) such as CreutzfeldtJakob disease (CJD) in man, bovine spongiform encephalopathy (BSE) in cattle and Scrapie in sheep. These diseases are characterised by an abnormally folded form of the host-encoded prion protein (PrP). The cellular, i.e. non-pathological isoform of PrP (PrPC) is present in most tissues and is most abundant in the central nervous system. Following infection, PrPC undergoes a conformational change during a posttranslational process, leading to altered physicochemical properties such as aggregation, insolubility and -sheet rich secondary structure. Sc This pathological isoform is designated PrP . Alzheimers disease (AD) is a progressive neurodegenerative disorder which is characterized by memory loss, confusion and a variety of cognitive disabilities. AD is the most prevalent dementia affecting nearly 2 % of the population in the western world, whereas the risk of AD increases dramatically in individuals beyond the age of 70. Neither a causal therapy for recovery nor a reliable early diagnosis, which could improve present therapeutically or preventional approaches, is yet available. Today, the most reliable diagnosis of AD is the post mortem identification of amyloid plaques and neurofibrillary tangles in the respective brain. The major component of the amyloid plaques is the Amyloid- (1-42) peptide (A). Within this project we focussed on the detection of PrP and A particles, because the according diseases are of particular interest as prion

FIG. 1: Surface-FIDA A) Scheme of surface-FIDA; B) Scanning scheme; C) Principal of particle counting: Fluorescence peak caused by the labelled aggregate.

The latest development of this test system is based on fluorescence microscopy using fluorescence intensity distribution analysis (FIDA). It is quantifying the number and size of aggregates simultaneously labelled by two different antibodies for dual colour FIDA [1]. Only aggregates and oligomers but not monomeric proteins are detected. To increase the sensitivity, particles were concentrated in the two dimensional space by immobilizing it to capture antibodies on the surface of the slide. Laser beams are scanning the surface systematically so that even single particles are detected [2, 3]. We successfully established surface-FIDA as a diagnostic tool for prion diseases [2]. The infectious agents of prion diseases are composed

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primarily of the pathogenic isoform of the prion Sc PrP . In contrast to its cellular isoform, the pathogenic isoform PrPSc forms insoluble aggregates. Hitherto accredited prion tests use the proteinase K (PK)-resistance of PrPSc as a marker for the disease. These prion tests offer only a limited sensitivity because of varying portions of disease related aggregated PrP which is not PKresistant. Therefore prion protein aggregate detection which does not rely on PK-digestion is favourable because it allows detection of both, PKSc resistant and PK-sensitive PrP , aggregates. We could successfully verify our novel test system for correct diagnosis of Scrapie infected hamsters as well as BSE-infected cattle in the clinical stages of diseases. Furthermore, we were able to detect PrP aggregates in the cerebrospinal fluid (CSF) of BSE-infected cattle for the first time [1] (Fig. 2). The use of three different antibodies (capture and two detection probes) but with overlapping epitopes delivered the best specificity within the assay [4].

FIG. 2: Detection of PrP aggregates in cerebrospinal fluid (CSF) of BSE-infected cattle by surface-FIDA Surface-FIDA of BSE-infected and healthy cattle CSF. In the histograms the mean values of burst number detected within the surface-FIDA measurements are shown. Different capture probe combinations of the antibodies D18, Saf32 and 12F10 are used. CSF samples from two different BSE-infected cattle (dark grey) and two different healthy controls (light grey) were analysed. The number of peaks varied independently of the labelling grade of the probes

FIG. 3: Detection of A aggregates in CSF of AD patients A) The sensitivity of the assay was determined by dilution of the aggregates in CSF. For all measurements (three measurements standard deviation), burst number detected by 2D-FIDA during 0.5 min are shown. Fluorescence labelled antibodies 6E10 and 19H11 were used as detection probes. B) 2D-surface-FIDA was applied to 20 l crude CSF of AD patients and control patients not affected by AD. Number of bursts (three measurements standard deviation) obtained by 2D-FIDA during 0.5 min are shown. [1] Birkmann E, Henke F, Weinmann N, Dumpitak C, Funke SA, Willbold D, Riesner D. (2007) Counting of single prion particles bound to a capture-antibody surface (surface-FIDA). Vet. Microbiol. 123, 294304. [2] Birkmann E, Schfer O, Weinmann N, Dumpitak C, Beekes M, Jackman R, Thorne L, Riesner D. (2006) Detection of prion particles in samples of BSE and scrapie by fluorescence correlation spectroscopy without proteinase K digestion. Biol. Chem. 387, 95102. [3] Funke SA, Birkmann E, Henke F, Grtz P, LangeAsschenfeldt C, Riesner D, Willbold D. (2007) Single particle detection of Abeta aggregates associated with Alzheimer's disease. Biochem. Biophys. Res. Commun., 364 (4), 902-907 [4] Birkmann E, Henke F, Funke SA, Bannach O, Riesner D, Willbold D. (2008) A highly sensitive diagnostic assay for aggregate-related diseases e.g. prion diseases and Alzheimers disease. Rejuvenation Res. 11(2), 359-363 [5] Funke SA, Birkmann E, Henke F, Grtz P, LangeAsschenfeldt C, Riesner D & Willbold D (2008) An ultra-sensitive assay for diagnosis of Alzheimers disease. Rejuvenation Res. 11(2), 315-318

We also established the surface-FIDA assay for the detection of Alzheimers A aggregates. We showed that the assay is sensitive enough to detect aggregates in the picomolar range. First measurements show a clear distinction between AD diseased people and non-demented controls by analysing CSF [3, 5] (Fig 3). During the next steps we will adapt the highly sensitive test system for diagnosis of human prion diseases like Creutzfeldt-Jakob disease and other aggregate related diseases, e.g. Parkinsons disease. Furthermore we will develop surfaceFIDA to an imaging assay.

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NMR structure of the M.Loti K1 channel cyclic nucleotide binding domain

S. Schnke
1 2 3 4 1,3

M. Stoldt

1,3

K. Novak , U. B. Kaupp
2

2,4

D. Willbold

1,3

ISB-3: Structural Biochemistry ISB-1: Cellular Biophysics Institute of Physical Biology, Heinrich-Heine-University, Dsseldorf Center for Advanced European Studies and Research (Caesar), Bonn

Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels, play crucial roles in neuronal excitability and signal transduction of sensory cells. HCN and CNG channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide-binding domain (CNBD). However, the mechanism by which the binding of cyclic nucleotides opens these channels, is not well understood. We report the solution structure of the isolated CNBD of a cyclic nucleotide-sensitive K+ channel from Mesorhizobium loti. The protein consists of a wide antiparallel -roll topped by a helical bundle comprising ve -helices and a short 310 -helix. In contrast to the dimeric arrangement (dimerof-dimers) in the crystal structure, the solution structure clearly shows a monomeric fold. The monomeric structure of the CNBD supports the hypothesis that the CNBDs transmit the binding signal to the channel pore independently of each other. Ion channels activated by cyclic nucleotides play key roles in neuronal excitability and signaling of visual and olfactory neurons. They belong to two subfamilies: Cyclic nucleotide-gated (CNG) channels, and hyperpolarization-activated and cyclic nucleotidegated (HCN) channels. Both channel types share a carboxy-terminal cyclic nucleotide-binding domain (CNBD). HCN channels are activated by hyperpolarization and their activity is modulated by cyclic nucleotides. In contrast, CNG channels are voltage independent and require cyclic nucleotides to open. Binding of cyclic nucleotides promotes the opening of the channel. Probably, a conformational change in the CNBD is propagated to the pore. Recently, a prokaryotic cyclic nucleotide-sensitive K+ -channel, designated MloK1, has been identied in Mesorhizobium loti. MloK1 harbors six transmembrane domains (S1-S6), a GYG signature sequence for K+ selectivity, and a conserved CNBD is connected via a short C-linker to S6 (Fig. 1). The longer C-linker (80 residues) of mammalian CNG channels is important for relaying the binding signal to the channel gate. Crystal structures of mammalian HCN channel CNBDs revealed that neighbouring C-linkers contribute virtually all contacts between subunits in the tetrameric protein. The crystal structure of the

isolated CNBD of MloK1 suggested that subunits are organized as dimers. The dimer interface formed by the short linker has been proposed to be involved in channel gating [1].

Subunit topology and assembly of the full-length MloK1 cyclic nucleotide-gated K+ channel. MloK1 consists of four subunits. Each subunit encompasses six transmembrane segments S1-S6 (yellow) and an intracellular CNBD (shown in ribbon representation).
FIG. 1:

However, an electron microscopy study of the complete channel reveals a four-fold symmetry of subunit arrangement [2]. The CNBDs appear as independent domains separated by discrete gaps, suggesting that CNBDs are not interacting with each other. Furthermore, the MloK1 channel and the isolated CNBD bind cAMP with similar afnity in a non-cooperative fashion [3]. Here we study the solution structure of the monomeric CNBD in complex with cAMP by nuclear magnetic resonance (NMR) spectroscopy (Fig. 2) and compare it with the structure in the dimer (Fig. 3).

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be set apart from its vertebrate cousins by the lack of a full-blown C-linker that coordinates intersubunit contacts. This conclusion is supported by an EM study of the complete MloK1 channel. An important feature of the EM structure is that the four CNBDs are separated by discrete gaps and that four isolated CNBDs could be modelled into the electron density map. This structure predicts that binding sites act independently. The C-linker contact observed in the crystal structure was possibly enforced by the packing of dimers in the crystal and that these contacts seem to be functionally irrelevant. In fact, cAMP and several analogues bind noncooperatively to the monomeric CNBD and the tetrameric full-length MloK1 with virtually identical high afnity [3]. Together with the presented solution structure, this shows that MloK1 cyclic nucleotide binding sites are functionally independent of each other [3, 5].

Solution structure of the isolated cyclic nucleotidebinding domain. A: Superposition of the backbone traces and all cAMP atoms of the family of 15 NMR structures with the lowest CYANA target function. Backbone atoms of the N- and C-terminal ends (residues Q216 to V218 and A351 to A355) are not shown and were not used for least-square superposition of the structures. B: Ribbon representation of the CNBD structure with the lowest target function. The cAMP ligand is shown as a stick model. Secondary structure elements are labelled.
FIG. 2:

The solution structure is very similar to the structure of a monomer in the dimer crystal [1]. The comparison of all CNBD backbone coordinates (residues V218 to G350) between solution and crystal structure results in a r.m.s. displacement value of 0.21 nm. However, the coordinates for the N-terminal residues (V218 to P241) which represent the 1-helix, 310 helix and associated loop regions differ remarkably. The 1-helix region (G221 to A231) in the solution structure is a straight helix without bending (Fig. 3). In the crystal structure, however, residues R220 to N226 of 1 are bent and form the dimer interface. The two most important insights of our study [4, 5] are that (1) the CNBD even at the high concentration required for NMR measurements is a monomer and that (2) the solution structure, except for the Nterminal C-linker region, is similar to the monomer structure in the dimer crystal structure. The much longer C-linker region of vertebrate CNG and HCN channels is involved in intra- and intersubunit contacts and contributes virtually all contacts between the subunits in the tetrameric crystal structures of CNBDs from two different HCN channels. The formation of dimers and tetramers from monomeric CNBDs requires cAMP, suggesting that rearrangement of the C-linker interface represents an important gating event. Thus, the MloK1 channel seems to

Comparison of NMR and crystal structure. A: Comparison of the solution structure (blue) and the crystal structure of the CNBD (light gray). Part of the second domain in the dimer crystal structure that is involved in the dimer interface is depicted in green. Alignment of the solution and crystal structure backbones (residues V218 to G350) yielded an r.m.s. displacement of 0.21 nm. A comparison of the solution and crystal structure excluding N-terminal residues (V218 to P241) results in an r.m.s. displacement value of 0.12 nm. B: CNBD backbone comparison of the monomeric solution structure (shown in blue) and the backbone of the crystal structure dimer (shown in red).
FIG. 3:

[1] G. M. Clayton, W. R. Silverman, L. Heginbotham and J. H. Morais-Cabral, Cell 118, 615-627 (2004). [2] P. L. Chiu, M. D. Pagel, J. Evans, H. T. Chou, X. Zeng, B. Gipson, H. Stahlberg, and C. M. Nimigean, Structure 15, 1053-1064 (2007). [3] A. Cukkemane, B. Gruter, K. Novak, T. Gensch, W. Bonigk, T. Gerharz, U. B. Kaupp, and R. Seifert, EMBO Rep. 8, 749-755 (2007). [4] S. Schnke, M. Stoldt, K. Novak, U. B. Kaupp, and D. Willbold, Biomol. NMR Assign. 1, 179-181 (2007). [5] S. Schnke, M. Stoldt, K. Novak, U. B. Kaupp, and D. Willbold, EMBO Rep., Manuscript Accepted (2009).

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Structural characterization of GABARAP-ligand interactions

Y. Thielmann1 , O.H. Weiergrber2 , J. Mohrlder1 , D. Willbold1
1 2

ISB-3: Structural Biochemistry ISB-2: Molecular Biophysics

The GABAA receptor-associated protein (GABARAP) plays an important role in intracellular trafcking of several proteins. It undergoes a C-terminal lipidation process that enables anchoring in the cytosolic leaet of cellular membranes. While the three-dimensional structure of GABARAP itself has been determined, structural investigation of complexes with its interaction partners has just commenced. Our recent work has established that GABARAP features two hydrophobic binding sites (hp1 and hp2), which play an essential role in complex formation with native ligands, such as calreticulin and the heavy chain of clathrin. Originally, GABARAP has been identied as a protein interacting with a cytoplasmic loop of the GABAA receptor 2 subunit. Colocalization with the GABAA receptor in cultured cortical neurons and the presence of a tubulin binding site qualied this protein as a potential adaptor linking the GABAA receptor to the cytoskeleton. GABARAP and its paralogues are members of the ubiquitin superfamily which is characterized by a so-called -grasp fold. The latter consists of a central four-stranded mixed -sheet with two helices located on its concave side. A hallmark of the GABARAP family is an N-terminal extension containing two additional -helices, which are attached to the convex face of the -sheet. Proteins of the GABARAP family are processed by a ubiquitin-type conjugation machinery, with the nal step involving covalent linkage to membrane lipids instead of proteins. The number of known GABARAP interaction partners is rising steadily. Current evidence indicates that it might play a major role in subcellular transport of various cytosolic and membrane-associated proteins. Phage display screening for GABARAP binding peptides indicated a high prevalence of tryptophan residues in potential ligands. Therefore, free indole and several derivatives were chosen as probes to characterize the binding properties of GABARAP by nuclear magnetic resonance (NMR) spectroscopy [1]. The specic behaviour of individual resonances indicated the presence of two apolar grooves on the surface of GABARAP which display different afnities for indole derivatives. If this binding site is biologically relevant, GABARAP interaction partners should display a surface-exposed conserved tryptophan. In1: Complex of GABARAP with a high-afnity ligand (K1). GABARAP is depicted as ribbon model with the -grasp domain and the N-terminal extension coloured in shades of blue. The peptide is shown with red and gray backbone; hydrophobic side chains involved in complex formation are indicated. FIG.

deed, such an invariant tryptophan can be found in the binding motifs of the GABAA receptor 2 subunit as well as additional physiological ligands, as discussed below. Among the GABARAP binding peptides identied by phage display screening, the "K1" sequence (DATYTWEHLAWP) was found numerous times in independent experiments. The interaction of this high-afnity ligand with GABARAP was studied using NMR spectroscopy and X-ray crystallography [2]. Although the K1 peptide makes contact with GABARAP in its entire length (Fig. 1), complex formation appears to be dominated by hydrophobic interactions mediated by two tryptophan residues (W6 , W11 ) and a leucine side chain (L9 ). It is important to note that ligand binding to GABARAP induces significant conformational changes in the protein. Combining pulldown experiments and data mining, the heavy chain of clathrin was identied as a physiological GABARAP binding partner. NMR titration of GABARAP with a 13-mer clathrin peptide revealed changes which are clearly indicative of a direct interaction. The biological signicance of the clathrinGABARAP interaction might relate to the GABAA receptor: endocytosis of the receptor is crucial for control of receptor numbers at the postsynaptic membrane of neurons. This process depends on the for-

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Proposed structure of the GABARAP-calreticulin complex. Globular domain and P domain of calreticulin are shown in light and dark red, respectively, the core binding motif (CRT178-188 ) in blue. GABARAP is displayed as surface representation, with colours indicating residues affected in NMR titrations with CRT178-188 (blue) and the entire P domain (red).
FIG. 2:

mation of coated pits and is mediated by clathrin. The interaction with clathrin heavy chain may therefore represent an important aspect of GABARAP function in GABAA receptor trafcking. Calreticulin is another GABARAP binding partner identied by a database search and veried using biochemical techniques [3]. NMR spectra of GABARAP in the presence of calreticulin showed clear indications of complex formation. Surface plasmon resonance experiments with calreticulin and GABARAP yielded a dissociation constant of 64 nM; this is the strongest interaction reported thus far for either protein. In a subsequent study, different calreticulin fragments were investigated for their interaction with GABARAP. These included the complete proline-rich (P) domain (aa 177-288) and a short segment thereof (aa 178-188) containing the putative GABARAP binding motif [4]. These data suggest that the peptide contains the core binding motif, but the adjacent domains of calreticulin account for additional contacts. The three-dimensional structure of the GABARAP-CRT178188 complex could be determined by X-ray crystallography. The ligand attains an elongated conformation in close contact to GABARAP, interacting with both hp1 and hp2. A structure model of full-length calreticulin reveals that its proposed GABARAP binding site is located at the N-terminal junction between the globular domain and the P domain (Fig. 2). The minimal machinery required to maintain membrane fusion in vivo includes NSF, a hexameric ATPase belonging to the AAA (ATPases associated with various cellular activities) class. GABARAP has been identied as an interaction partner of NSF, which modulates the subcellular localization of the ATPase. Whereas crystal structures have been determined for single domains of the NSF protomer, a structure of the full-length polypeptide is still lacking. Therefore, a homology model of the entire NSF complex was generated, using the related ATPase p97/VCP as a template [5]. Docking experiments with GABARAP

Model of hexameric NSF with docked GABARAP molecules. NSF subunits are shown in blue and light grey, GABARAP in orange. See text for details.
FIG. 3:

resulted in the model shown in Fig. 3. The proposed NSF-GABARAP interface is composed of an apolar core, which is anked by polar contacts. An interesting aspect of the complex model is the proximity of GABARAP to the nucleotide binding site in the major ATPase domain of NSF, suggesting a regulatory impact on ATP binding and/or hydrolysis. Due to its C-terminal lipidation, binding of GABARAP to NSF in this conguration could serve to anchor the complex to membranes. These investigations have focused on the characterization of GABARAP interactions with articial as well as physiological ligands, applying a wide repertoire of methods from molecular and cellular biology, biochemistry and structural biology. Together, our data have provided novel insight into structural foundations and dynamic properties of protein-protein interactions of GABARAP and its homologues, which ultimately govern the biological functions of this novel class of adaptor molecules.

[1] Y. Thielmann, J. Mohrlder, B. W. Koenig, T. Stangler, R. Hartmann, K. Becker, H.-D. Hltje and D. Willbold, ChemBioChem 9, 17671775 (2008). [2] O. H. Weiergrber, T. Stangler, Y. Thielmann, J. Mohrlder, K. Wiesehan and D. Willbold, J. Mol. Biol. 381, 13201331 (2008). [3] J. Mohrlder, T. Stangler, Y. Hoffmann, K. Wiesehan, A. Mataruga and D. Willbold, FEBS J. 274, 5543 5555 (2007). [4] Y. Thielmann, O. H. Weiergrber, J. Mohrlder and D. Willbold, FEBS J. 276, 11401152 (2009). [5] Y. Thielmann, O. H. Weiergrber and D. Willbold, Proteins (in revision).

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104

IBN-2: Bioelectronics
Director: Prof. Andreas Offenhusser

The interfacing of man-made electronics with bio-systems like DNA, redox proteins, enzymes and cells not only allows us to learn about molecular processes in biology, but also paves the way to using it in derived sensory devices. Some of these have already had a profound impact on clinical diagnostics. Technological approaches also can be inspired by biological systems potentially leading to new cognitive and sensory approaches to information processing. Our research aims to develop bioelectronic devices that combine biological systems-from single biomolecules to living cells and organisms-with electronics. Within the broad field of bioelectronics, we have identified two domains where our expertise in nanotechnology can add value for improving the efficiency and sustainability of current environmental research and health care. Sensing and imaging Electronic devices that detect trace amounts of biochemicals in the environment or in bodily fluids will allow far earlier detection than current technologies and will therefore facilitate appropriate reactions. Magnetic sensors have evolved due to the ever-increasing need for improved sensitivity. Ultra-sensitive superconducting quantum interference devices (SQUIDs) have a great potential for biomedical sensing. Microwave to terahertz sensing techniques, based on collectively vibrational modes of complex molecules at terahertz frequencies, relaxation of the dielectric function

in the micro- and millimetre wave range, and ionic conductivity at radio frequencies, have great potential for applications in biology, medicine, airline, and public security. Bioelectronic devices and biomedical applications The use of biomolecules as the building blocks of higher-level functional devices will lead to applications ranging from the integration of biomaterials with electronics in recognition to sensing devices, such as biosensors. Bioelectronics research also exploits the use of biomolecules to perform electronic functions that semiconductor devices currently perform, thereby offering the potential to increase integration in combination with additional functionalities at the nanometer level. Living cells and tissues exhibit an extraordinary range of functions including highly selective biochemical sensing (even in chemically noisy environments), protein synthesis, and information processing. Functional interfaces between neurons and micro-/nanodevices will have the potential to enhance in-vitro applications ranging from basic neuroscience research and disease modelling to drug screening and biosensors. Future in-vivo applications of bioelectronic devices include, for example, stimulating and recording deep-brain activity, managing pain, and restoring damaged nervous pathways.

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A Model for the Cell-Sensor Interface and Comparison with Experiment

M. Pabst1,2, G. Wrobel1,2, F. Sommerhage1,2, S. Ingebrandt1,2, A. Offenhusser1,2
1 2

IBN-2: Bioelectronics JARA-FIT

Electrogenic cells are able to generate electrical signals which can be measured by various invasive electrophysiological methods such as patch-clamp or sharp microelectrode recordings. Growing cells on the surfaces of e. g. metal microeclectrodes or field-effect transistors allows the recording of an extracellular component of these signals. For an understanding of such extracellular signals it is mandatory to get detailed topographical as well as electrical information about the cell-sensor interface. In a first approximation, this interface can be described by a flat disk between cell membrane and sensor surface. For a correct description of the signals, the electrodiffusion of ions in this interface is modeled by using the stationary Poisson-Nernst-Planck equations. We solve the equations analytically, and derive expressions for the potential, the ionic charge densities, and the seal resistance. The results provide a method for determining the distance h between sensor surface and cell membrane. For human embryonic kidney cells, we receive h 70 nm. Comparison with transmissions electron microscopy studies shows reasonable agreement.

These changes in local extracellular voltage result from the flux of ions across the membrane of the cell, mainly sodium (Na+), potassium (K+), and calcium ions (Ca2+). The distribution and strength of the potential near the sensor depends on the geometry of the cleft between attached cell and sensor surface and the specific electrical behavior of the cell membrane. For a quantitative understanding of the extracellular signals it is necessary to describe the experimental situation. A schematic picture of a typical setup is shown in Fig. 1. Biological cells (here: human embryonic kidney (HEK293) cells expressing a voltage gated K+ channel) are cultured on top of the gate of a FET [1]. Outside the cell and inside the cleft, the region between cell and sensor surface, there is extracellular electrolyte solution. By electrical excitation, the ion channels in the cells membrane open and ions can flow from the inside to the outside. While in the upper part of the cell (free membrane) these ions just enter the surrounding electrolyte bath directly, it is different at the attached membrane. Here, the ions have to pass the cleft before entering the bath. The flux of ions into the cleft causes an increase of charge. Due to the connected electric field other charged ions will move: ions with similar charge leave the cleft and move into the surrounding bath while ions of opposite charge are attracted and enter the cleft. The change of charge inside the cleft influences the source drain current under the gate and is sensed by the FET. It can be directly correlated to the membrane current into the cleft.

FIG. 1: Schematic drawing of the cell-sensor interface between a HEK293 and a field-effect transistor (not to scale). The cell is located on the top of a FET gate. The cell is approximated by a half-sphere with a radius a = 15 m. The cleft height is h < 100 nm [1,2]. The gate area of 2 the FET is about 50 m . Smaller letter-size corresponds + to smaller a concentration of K .

The electrical activity of biological cells in vitro can be recorded by measuring changes in the local extracellular voltage with planar metal electrodes or integrated planar field effect transistors (FETs).

FIG. 2: Exemplary electrical coupling of a HEK293 cells + transfected with K channels to a FET gate. A) Voltage+ clamp stimulation pulse, B) transmembrane K current measured by patch-clamp, and C) corresponding VFET recording.

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Fig. 2 shows the comparison of whole-cell membrane currents (IM) and extracellular signal shapes (VFET) recorded with a p-channel FET for + HEK293 cells transfected with a K channel: (A) Rectangular stimulation pulses from the holding voltage led to (B) whole-cell current IM and (C) corresponding FET signals VFET for activation of the + K channel. The FET signal shape resembles a combination of different signal components, each with distinct kinetics depending on the respective cell type: (i) capacitive transients, caused by the capacitive coupling of the stimulation pulse, (ii) an increase of VFET to a steady-state amplitude, (iii) a partially instantaneously decline of VFET at the end the pulse, and (iv) a slow relaxation of VFET, that is absent in IM. For the description of the FET signal shapes the ion flux in the cleft has to be understood. In our approach we apply the Poisson-Nernst-Planck (PNP) equation system, a differential equation system consisting out of the continuity equation, the Nernst-Planck equation and the Poisson equation [2]. The system describes the potential and charge densities inside the cleft. By adapting the PNP system to our experimental situation, the system is reduced to an ordinary inhomogeneous Bessel differential equation. This equation can be solved analytically in the stationary case corresponding to the steady state pulse situation after 100 ms in Fig. 2. As a result we receive for the potential (r)
( r ) 1
r2 L2 +4 D 2 a a2 I 0 ( r / LD ) I ( a / L ) 1 D 0

+ solution, diffusion constant of K ions and the height h of the cleft (Fig.1). By correlating VFET as a function of the total cell current IM in steady state case for many cells, the last two formulae allow the calculation of an average height h. From Fig. 3 we receive for RJ = 0.57 M and in consequence 70 nm for the height h.

FIG. 3: Comparison of the steady-state p-channel FET signals (VFET) in dependence on the current through the attached membrane (IJM) for 10 cells.

Here r is is the radial position inside the cleft, a the radius, LD the Debye-length and I0 the Bessel -5 function. While LD/a is of the order 10 , the second term can practically neglected giving a parabolic potential, which corresponds to a constant total charge density inside the cleft. From the potential the charge densities can be derived showing a similar parabolic form like the potential. With the above results an expression for the measured voltage signal VFET of the FET in the steady state case can be derived (Fig. 2):

To compare our theoretical result for the height h, experiments have been made to measure the height directly [3]. Transmission electron microscopy (TEM) was applied to analyze the cellsensor interface. The studies were complemented by imaging ellipsometry. As a result, it was found for HEK293 cells, that the contact geometry between cell membrane and substrate was dependent on the protein coating used for the substrate. In presence of polylysine, the measured average height h was in the range 35 40 nm, which is about half of the theoretical value. Considering the experimental uncertainties for growing cells on substrate, this is a reasonable agreement.

VFET = (1 + )RJ I JM
with the cleft resistance RJ and the ion current IJM of the cell into the cleft. The correction term takes into account the surface properties of the metal gate of the FET underneath the cell. It turns out that is of the order of 1, which means half of the signal comes from the gate properties and the other half from the membrane current into the cleft. RJ is given by

[1] G. Wrobel, R. Seifert, S. Ingebrandt, J. Enderlein, H. Ecken, A. Baumann, U. B. Kaupp, A. Offenhusser, Cell-transistor coupling: Investigation of potassium currents recorded with p- and nchannel FETs, Biophys. J. 89, 3628-3638 (2005) [2] M. Pabst, G. Wrobel, S. Ingebrandt, F. Sommerhage, A. Offenhusser, Solution of the Poisson-Nerst-Planck equations in the cellsubstrate interface, Eur. Phys. J. E. Soft Mat. 24, 1-8 (2007) [3] G. Wrobel, M. Hller, S. Ingebrandt, S. Dieluweit, F. Sommerhage, H. P. Bochem, A. Offenhusser, Transmission electron microscopy study of the cellsensor interface, J. Roy. Soc. Interf. 5 (2008).

RJ = q
B

k BT 2 B 8e0 ntot hDK

with q, kB, T, e0, n tot, DK, h being a correction factor close to 1 due to the experimental situation, the Boltzmann constant, temperature, elementary charge, ion density of the surrounding electrolyte

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Nanostructured Interfaces for Bioelectronic Systems

B. Wolfrum1,2, Y. Mourzina1,2, F. Sommerhage1,2, B. Hofmann1,2, D. Brggemann1,2, A. Offenhusser1,2
1 2

IBN-2: Bioelectronics JARA-FIT

The properties of a bioelectronic interface (interface between an individual cell and a chip transducer) are of primary importance for bioelectronic systems. Nanopatterned surfaces promise to enhance the interface quality regarding to electronic signal transduction, cell adhesion, and selectivity. Electrode materials with increased surface area have the best electrode properties due to a large effective area, favouring a smaller electrode resistance, a larger capacitance, and further decreasing of the feature size. This reduces the noise level and increases the current injection capability. Here, we present a concept of a large-scale patterned gold nanopillar array interface on semiconductor substrates with the aim of functional coupling to electrogenic cells. Nanostructuring is based on the combination of an imprint approach with a nanotemplate-assisted electrodeposition. This new and versatile method can be adopted for different interface materials of electronic circuits and their coupling to both cellular and molecular sensing components.

Interfaces with nanoscopic sizes play a vital role in electrochemistry, catalysis, energy storage/conversion devices, cell adhesion studies, bioelectronics, and information technology. Intensive research is focused on patterned nanopillar (NPA) array interfaces of diverse materials. Thereby, the specific and precise positioning of nanofeatures on a large scale is especially challenging. Fabrication methods of NPAs can be divided into direct lithographic methods utilizing e-beam or focused ion beam (FIB) writing, nanoparticle growth or deposition by self organization, and template-assisted methods. While the direct lithographic methods offer the most versatility they are usually limited to small area patterning due to speed limitations caused by the sequential processing. Methods based on self organization can be applied on a larger scale but often lack the versatility obtained by the direct methods. Furthermore, most fabrication methods based on self organization without template assistance are limited to specific materials. We therefore combined an imprint approach with a nanotemplate-assisted electrodeposition to create large-scale micro- and nanometer sized arrays of gold nanopillars at predefined positions on a semiconductor substrate. Imprint techniques are often preferred over other direct patterning techniques, because lithography has to be used only for the fabrication of the stamps.

FIG. 1: Process sketch for the fabrication of the largescale patterned arrays of Au-nanopillars.

FIG. 2: SEM images of silicon stamps for imprinting aluminum layers (a-d). Top and side view of dotted FZJ (a,b) and line patterns (c,d) directly after RIE (a), at different stages of KOH etching (b,c), and after removal of the silicon oxide mask (d). The line width at the stamp tip is 130 nm.

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The process for the fabrication of patterned nanopillar arrays is shown in Fig. 1. The Si stamps for the indentation of the aluminum surface (a) are created by lithography combined with anisotropic wet chemical etching of silicon [1]. The stamp patterns have different geometries, Fig. 2, and cover an area of 4x4 mm on an 11x11 mm chip, the smallest initial feature size of the stamp being 1 m. The pattern of the stamp is transferred via imprinting onto a Ti/Au/Al covered substrate (b). Here, we show that an imprint approach on thin aluminum films can be used to define nano- and mircopatterned regions of pores on a substrate, which selectively act as a template for the electrodeposition of nanopillars.

the Au-nanopillars depend on anodization conditions of the aluminum film and the subsequent pore enlargement in phosphoric acid. For example, we fabricated nanopillar arrays with a diameter down to 20 nm and a pillar to pillar distance of ~50 nm by anodizing the aluminum film at 22 V in sulfuric acid (3%). Similar arrays could be used for surface enhanced Raman scattering [3] and cell adhesion studies [4]. Figure 3(c-f) shows images of patterned Au-nanopillar line arrays. The minimum interspace distance of the patterned nanopillar lines is determined by the resolution of the optical lithography used for the stamp fabrication. Smaller interspace distances might be achieved by performing multiple imprints with the same stamp using a precise positioning system.

FIG. 4: SEM image of a rat neuronal cell growing on a gold nanopillar surface (a) and a microelectrode active area modified with gold nanopillar pattern (b). FIG. 3: FZJ-silicon stamp pattern (a) and the reproduction of the stamp pattern as Au nanopillar arrays (b). The inset shows an image enlargement of a single dot feature of (b). Images (c-f) show top and side views of Au nanopillar fences created by using line stamps to indent the aluminum film. The lines extend up to the stamp size of 4 mm.

Patterned aluminum films are subsequently anodized in 0.3 M oxalic acid at 3C and 40 V to obtain nanoporous alumina membrane (AM) template with an approximate interpore distance of 80 nm (c) [2]. The anodization is stopped after the nanochannels originating from the indented surface reach the underlying gold substrate. The barrier layer, which possibly remains at the bottom of the pores originat ing from the predefined regions, is dissolved in 5% phosphoric acid. AM acts as a template material for the metal deposition, in which gold is exclusively formed inside the nanochannels of the porous alumina. The template material can be selectively etched in alkaline solution to expose the gold nanopillars arrays (d). In Fig. 3 it is shown how a large-area stamp pattern (Fig. 3a) exhibiting dots of different interspaces can be reproduced with pads of gold nanopillar arrays (Fig. 3b). Each nanopillar array is composed of approximately 60 Au-pillars with a width and height of ~35 and ~300 nm, respectively. The interspace distance and width of

We found that the nanostructured surfaces presented here were biocompatible with cell lines expressing ion channels as well as primary neuronal cells from the rat cortex, Fig. 4a. We further showed that the nanotemplate-assisted electrodeposition can be combined with a prepatterned electrode array as a precursor for NP growth to create a new type of a 3-D nanostructured interface for coupling with electrogenic cells, Fig. 4b. We propose that this approach can be extended to different interface materials of elec tronic circuits and their coupling to both cellular and molecular sensing components. Acknowledgements: A. Steffen, J. Mller, H.-P. Bochem, M. Nonn, M. Prmpers, Dr. M. Lepsa, D. Schwaab, Dr. D. Mayer, Dr. S. Ingebrandt, A. Fox, S. Schaal, B. Hermanns.
[1] B. Wolfrum, Y. Mourzina, D. Mayer, D. D. Schwaab, A. Offenhusser, Small 2 (2006) 1256. B. Wolfrum, Y. Mourzina, F. Sommerhage, A. Offenhusser, Nano Letters 6 (2006) 453. G. Sauer et al., J. Appl. Phys. 2005, 97, 024308. N. Walter, C. Selhuber, H. Kessler, J. P. Spatz, Nano Lett. 6 (2006) 398.

[2]

[3] [4]

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Functional Networks of Rat Neurons on in-situ Patterned KDI

S. Meffert1,2, K. Adamiak3, R. Helpenstein1,2, B. Hofmann1,2, A. Offenhusser1,2
1 2

IBN-2: Bioelectronics JARA-FIT 3 DWI, RWTH Aachen University

This study aimed to increase the lifetime and stability of in vitro network of neurons. We tested KDI, a 12 amino acid peptide of laminin peptide, using in situ-microcontact printing to create a chemically attached peptide pattern on silicon oxide substrate. By atomic force microscopy (AFM) a pattern thickness of about 2.9 nm which corresponds to a peptide monolayer could be measured. Rat cortical neurons were seeded on the KDI pattern and tested on their viability and functionality. Synaptically connected neurons were confirmed by double patch-clamp recording and immunostaining experiments. The expression of the pre- and postsynaptic proteins vesicular GABA transporter (VGAT) and gephrin could also be evidenced. Our data indicated that a monolayer of in situ-printed KDI peptide is sufficient for neurons adhesion and also for formation of functional and stabilized networks. By future application of these patterns as growth substrate on sensor arrays the extracellular recording of single neurons should be further improved. There is an increasing need in the field of neuroelectronic devices to immobilize functional proteins on the device surfaces for coupling extracellular signals from neuronal networks and long-term recording. Immobilization on surfaces can be easily accomplished by direct adsorption. However this method results partially thick and inhomogenous protein layers, denaturation of the protein, as well as unstable attachment affecting neurons and network function. Covalent attachment of functional proteins or peptides are one solution and are important when a coated substrate is subjected to a flowing solution or exposed for a long period of time in solution. In this study we aimed to construct a 1) chemically stable, 2) reproducible, 3) thin as possible growth pattern enabling long time stability and functionality of neuronal networks on silicon oxide substrates. Thereby, we used the in situmicrocontact printing (CP) which was recently established to print proteins/peptides without inducing their denaturation [1]. In situ CP was

combined with a protocol for covalently attaching peptides to construct a chemically stable peptide pattern of KDI, a 12 AS consisting synthetic peptide deriving from neurite outgrowth-promoting domain of laminin. KDI (cystein-terminated KDI peptide, 5 M) was printed on aminosilanized (APTES, 3-aminopropyl-trietoxy silane) and crosslinker (sulfo--maleimidobutyryloxysuccimide ester) attached glass substrate. To investigate the fidelity of the pattern, carboxyfluorescein labelledKDI was used for one set of experiments. Printed KDI patterns were first analyzed by phase contrast and fluorescence microscopy. Fluorescence images confirmed the transfer of carboxyfluorescein labelled KDI on glass substrate and illustrated that the pattern was almost without any interruptions and a high degree of uniformity.

FIG. 1: AFM analysis was performed using Nanoscope IV Multimode Instrument with a Nanoscope IV controller and a 15 m scanner in tapping mode. The KDI in-situ imprint shows a smooth topography of the printed pattern (A) and a section analysis with a small roughness of about 1nm (A, right). Detailed analysis (B) revealed a layer thickness of 2.868 nm (B, right).

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In contrast, no KDI pattern was observable by phase-contrast microscopy which indicates a reduced thickness of this peptide pattern in comparison to the standardly printed protein mix PECM (polylysine/ extracellular matrix gel) [2, 3]. For further characterization of the peptide imprints and measuring the layer thickness atomic force microscopy (AFM) was carried out using a Nanoscope IV Multimode Instrument with a Nanoscope iV controller and a 15 m scanner. Samples were scanned in tapping mode with resonance frequencies between 260 and 300 kHz. The KDI-imprint showed a smooth topography of the pattern indicating a homogenous peptide coupling to the surface. Further AFM analysis of one section confirmed the slight layer roughness and revealed a thickness of 2.9 nm (Fig.1) which was in the range of the theoretical calculation of a peptide monolayer [4-5]. The KDI pattern was subsequently tested in cell culture on neurons functionality and network stability.

guidance of the processes along the lines were achieved with high compliance. The neurons developed a mature morphology and differentiated to network forming neurons. Apart from the morphological evidence the neuronal identity and functionality was confirmed by immunostainings and electrophysiological experiments. Single and double patch clamp experiments elicted spiking neurons and synaptically connected neurons grown on the KDI pattern. The cortical neurons were stained with antibodies against the pre- and postsynaptic proteins, vesicular GABA transporter (VGAT) and gephrin. The expression of both proteins could be identified at DIV 7 and DIV 12. Finally, the network formed on KDI pattern were stable without detaching from the substrate up to DIV 22. In summary, we were able to construct a chemically stable, homogenous, 2.9 nm-thin KDI and likely monomolecular peptide layer. The peptide layer was tested to be suitable as substrate for adhesion and outgrowing of neurons confined to the pattern geometry. This peptide pattern could further be proved to be suitable for long-time culturing of neurons and due to the stability of networks in cell culture. With these data, we propose that KDI patterned substrate may be useful for extracellular recordings to improve the signal coupling by reducing the celldevice distance. We thank D. Mayer and S. Gilles for AFM measurements. Financial support came from the Sony Deutschland GmbH.
[1] D. Schwaab, Dissertation at the RWTH Aachen (2007). [2] A. Vogt, L. Lauer, W. Knoll, A. Offenhusser, Biotechnol. Prog. 19, 1562 (2003)

FIG. 2: Phase contrast image of rat cortical neurons grown on KDI pattern DIV 7, forming geometrically defined networks (A). Patterned neurons were fixed and immunostained with antibodies against the pre- and postsynaptic proteins VGAT (red) and gephyrin (green). Laser scanning images of neurons grown on KDI immunostained against VGAT and gephrin showing high expression of both synaptic proteins DIV 7 (B) and DIV 12 (C).

[3] S. Bcker-Meffert, T. Decker, S. Schfer, A. Offenhusser, PB-10, 5th Int. Symp. Biomimetic Materials Processing (BMMP-5), 2005 [4] M. Scholl, C. Sprssler, M. Denyer, M. Krause, K. Nakajima, A. Maelicke, W. Knoll, A. Offenhusser, J. Neurosci. Methods 104, 65 (2000) [5] K. Adamiak, B. Hofmann, S. Bcker-Meffert, A. Offenhusser, 7th Gttingen Meeting oft he German Neurosciences Society, Germany, 738-5B (2007).

Cortical neurons from embryonic (E18) rats were prepared in a chemically controlled medium and seeded (16.000 cells/ cm2) [2] at the KDI patterned glass substrate. The substrates were optically controlled due to adhesion and outgrowth of the cells on the pattern. Figure 2 illustrates the behaviour of the neurons after 7 and 12 days in vitro (DIV7). The aligning of the cell bodies onto the 12m-nodes of the grid pattern as well as the

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Generation of protein gradients by nanoscale patterning

D. Schwaab1,2, P. Zentis1,2, S. Vlker1,2, S. Meffert1,2, D. Mayer1,2, A. Offenhusser1,2
1 2

IBN-2: Bioelectronics JARA-FIT

Combining high resolution lithography with microcontact-printing by means of hard plastomers it was possible to adjust the size of elements of a protein pattern and simultaneously the distance between them with sub 100 nm resolution. Rat neurons adhered onto these sub 10nm protein layers, apparently integrate over a large number of pattern elements and recognise the pattern as quasi homogeneous. However, the neurites showed pre-dominantly an aligned outgrowth corresponding to the underlying pattern. Thus, the technique proposed in this work maybe path the way to systemically study the influence of nanometer sized purely biochemical gradients on guiding neurons. These discontinuous gradients will further enable to evaluate the role of ECM/ligand spacing and in parallel the effect of defined gradient parameters on axon guiding. In developing nervous system the path of axons to reach their targets and establish neural circuits is directed by soluble and surface-bound biochemical gradients. Ligands of these gradients are detected by axonal growth cones which probe their environment by extending or retracting their filopodia. Although considerable effort has been directed towards characterizing chemotactic molecules and their receptors, the cellular mechanism by which neuronal growth cones sense these gradients remains generally unknown. It is envisioned to fabricate chemical gradients with length scales relevant to the biological involved cellular structures. The fabrication of continuous surface-bound gradients has been realized by a wide variety of methods [1]. Recently, von Phillipsborn et al. [2] used microcontact-printing to produce geometrically defined discrete gradients consisting of protein-covered spots varying in sizes and spacing. To transfer the concept of discrete gradients to the nanoscale dimensions of extracellular matrix fibrils nanostructuring techniques needs to be adapted. In the scope of this work we have developed a procedure to fabricate discrete purely biochemical gradients of extracellular matrix proteins by means of microcontact printing (CP). This technique directly transfers molecular inks from a polymer stamp to the target surface [3]. Here we show the transfer of ultra thin (10 nm or less) compact protein layers with sub 100 nm lateral resolution and demonstrate

the use of these films to guide neuronal outgrowth. Polyolefine plastomers (POP) were chosen as stamp material with a Youngs modulus of E=80 MPa (Avinity VP, Ticona). The high stiffness prevents the material from disrupting effects like sagging during printing process and facilitates high pattern fidelity. The plastomer stamps were structured by hot embossing by using a mold made of silicon. The molds contained a pattern with lines and spaces with both constant and varying widths ranging from 1 m to 75 nm, resulting in a line pattern with different pitches. A mixture of FITCpoly-D-lysine extracellular matrix proteins and (FITC-PDL/ECM) was used as ink. The stamps were dried with a nitrogen stream and pressed onto glass or silicon oxide substrate which was prehydrophilized by oxygen plasma. An uniform transfer of proteins on large length scales was verified by optical fluorescence microscopy (Fig. 1a), indicating also a high pattern fidelity. Images recorded at higher magnification by means of scanning electron microscopy (SEM) did not reveal any major pattern defects. Well-defined protein lines were observed as dark stripes in the SEM images (Fig.1b) with inverted contrast compared to fluorescence images. In the SEM images neither hints of disruptive effects like sagging or pattern collapse nor contrast variation over the imaged area were observed.

FIG. 1: Large scale images of the transfer of FITCPDL/ ECM ink to a SiO2. (A) Fluorescence microscopy image of the FITC dye coupled to PDL (excitation 488nm, emission 518nm) indicates schematically the area of the SEM image, see right. (B) SEM image at a magnification of 5450 with inverted contrast compared to (A), the dark

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The homogeneity of the protein coverage was further investigated with nanoscale resolution using Atomic Force Microscopy (AFM). The AFM images revealed that all structural elements of the pattern were transferred from the stamp to the hydrophilized SiO2 surface, even the 75 nm lines. An analysis of the line width and the deviation between designed pattern and printed features for most structures was smaller 10 % in average. The height of printed protein layers varied between 2 nm and 10 nm. To evaluate the response of neuronal cells to the nanoscale discrete protein gradients described above we analyzed the adhesion and outgrowth of primary neurons on these patterns by SEM. The isolated neurons were obtained from 18 day-old rat embryos (E 18) as described by Vogt et al. [4]. The cells were plated onto patterned SiO2 substrates. After 5-7 days in culture the cells were fixed, critical point dried and coated with a thin layer of platin/iridium.

FIG. 2: SEM image of a patterend SiO2 substrate with outgrown neurites of rat neurons. The neurites grow in alignement to the pattern of FITC-PDL/ ECM. On the left side of the image the nano pattern design was superimposed on the original SEM image to highlight that the filipodia attached also to the smallest elements of the pattern.

The cells adhered to the homogeneously coated and patterned areas with no differences due to the fact that even the largest elements of the pattern (1 m x 5 m) were much smaller than 2 the adhesive area (120 m ) of the soma size of cortical neurons. Furthermore, the SEM images revealed that the neurons extended neurites > 100 m which also arborized on both particular areas of the substrate. In contrast, clear differences were observed for the growth of neurites on the patterned area compared to the homogeneously coated one. On the patterned surface, neurites showed predominantly an aligned outgrowth corresponding to the underlying pattern indicating that the structure elements of the gradient were recognized in both main directions of the nanopattern which are perpendicular to each other. On the homogeneously coated surface the neurites formed an extensive meshwork of stochastically criss-crossing neurites. For a better analysis of the effect of nanopatterns on the neurite growth SEM images with a higher magnification were recorded. Imaging parameter were selected such (voltage 0.2 kV, inlens detector) that both protein pattern and neurons with outgrown neurites are displayed simultaneously (Fig.2). However the shallow protein layers (2 10 nm) were buried underneath the sputtered iridium layer, which results in a rather poor contrast and small protein features are difficult to reveal at some places. Therefore, the designed pattern was superimposed onto the SEM images aligned to the protein pattern underneath in order to highlight the areas which are covered with proteins. The SEM images further confirmed the alignment of the neurite growth with the pattern, even though a few neurites do not follow the underlying pattern. Growth cones could also be imaged demonstrating that the filipodia adhere at different structure elements including the smallest ones with a width of 75 nm. Thus, the technique proposed in this work may path the way to systemically study the influence of nanometer sized purely biochemical gradients on guiding neuronal cells by directing neurite outgrowth. These discontinuous gradients will further enables to evaluate the role of ECM ligand spacing and in parallel also the effect of defined gradient parameters on axon guiding.
[1] K. Dertinger, X. Jiang, Z. Li, V. Murthy, G. Whitesides, PNAS 2002, 1, 12542. [2] A. von Philipsborn, S. Lang, J. Loeschinger, A. Bernard, C. David, D. Lehnert, F. Bonhoeffer, M. Bastmeyer, Development 2006, 133, 2487 [3] A. Offenhusser, S. Bcker-Meffert, T. Decker, R. Helpenstein, P. Gasteier, J. Groll, M. Mller, A. Reska, S. Schfer, P. Schulte, A. Vogt-Eisele, Soft Matter, 2007, 3, 290 [4] A. Vogt, F. Stefani, A. Best, G. Nelles, A. Yasuda, W. Knoll, A. Offenhusser, J. Neurosci. Meth. 2004, 134, 191.

Figure 2 shows a SEM image of a SiO2 substrate after printing of FITC-PDL/ ECM and culturing cortical neurons for 7 days. The image was recorded at the transition from a nanopatterned area to an area homogeneously covered with proteins. The cells adhered to both areas however with distinct differences regarding neuronal outgrowth (data not shown). Averaged over the whole pattern the coverage of the surface with proteins was estimated to 45 % which is consistent with the particular pattern design. Normalized to the coverage with proteins, there was no obvious difference in the number of cells adhered on nanopatterned area (64 5) compared to the area of the surface that were homogeneously covered with proteins 124 29.

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114

IBN-4: Biomechanics
Director: Prof. Rudolf Merkel

Our research is directed towards a basic understanding of the physical principles, underlying structure and function of living cells. In this vast field we focus on the mesoscopic length scale from molecules to cells where principles of soft matter physics shape biological processes. On this length scale we study mechanical functions and properties of living cells, molecular aggregates, and molecules.

To achieve our goal, we perform complementary experiments on living cells and biomimetic model systems. By studying living cells we gain insight into the physiological relevance of processes and their likely mechanisms. At present, adhesion, locomotion, force generation, and mechanosensing of living animal cells are at the center of our interest. Further, experiments on model systems designed to mimic bioadhesion and the cytoskeleton enable us to achieve a quantitative understanding. In order to pursue this double strategy we develop and apply advanced methods for measurements on living cells and biomolecules as well as preparation and analysis techniques for tailored biomimetic systems. Our research strategy traverses the boundaries between physics, chemistry, and biology. Therefore researchers from all three classical sciences form the institutes scientific staff. Interdisciplinary cooperation on common projects on an everyday basis is one of the major strengths of the institute.

115

Deformation of Soft Solid Objects: Resolution Limit in Replica Molding

O.D. Gordan1 , Bo N.J. Persson2 , C.M. Cesa1,* , D. Mayer3 , B. Hoffmann1 , S. Dieluweit1 , R. Merkel1
1 2 3 *

IBN-4: Biomechanics IFF-1: Quantum Theory of Materials IBN-2: Bioelectronics now at Philips Research Laboratories, Care & Health Applications, Eindhoven - The Netherlands

The shape of liquid droplets results from the interplay between surface tension and external forces. In a similar way, very soft solids are affected by surface tension at scales relevant for micro-technology and biology. The characteristic smoothing length is proportional to the ratio between the surface tension and Young modulus of the material. This can be understood from the following qualitative argument: the elastic energy stored in a solid object, with linear size D and with uniform strain of order unity, scales with the volume of the solid as ED3 where E is the elastic modulus. The surface free energy scales with the surface area as D2 , where is the surface free energy per unit area. Thus one may expect a strong shape deformation if D is of order /E [1]. As an example, for very soft rubber E may be of order 10 kPa and if is of order 10 mJ/m2 we get D 1 m. Thus, soft micrometer sized solid objects may undergo shape deformations corresponding to a strain of order unity. This qualitative conclusion has many practical implications, e.g., for microtechnology and biology. Therefore nano and micromolding using elastic material will produce replicas with a characteristic smoothing length scale of /E, as can be seen in Figure 1. The experiments were performed using silicone rubber prepared using a two part kit, Sylgard 184, purchased from Dow Corning (Midland, MI). By adjusting the mixing ratio of the base (vinyl terminated polydimethylsiloxane) to the curing agent (methylhydrosiloxane-dimethylsiloxane copolymer) in the range from 10:1 to 55:1 the elasticity of the resulting rubber can be tunned between 2 MPa to 10 kPa. The mixtures were poured over silicon oxide molds patterned by conventional optical lithographic techniques. A glass coverslip was placed over the PDMS layer, and the whole ensemble was cured over night at 60 C. Before the molding process the PDMS was degassed in a desiccator using a mechanical pump. Upon curing the mixture undergoes a cross-linking reaction which will produce a solid elastomer. The elasticity of the resulting rubber depends on the cross-link density via the mixing ratio of the two constituents. In order to prevent PDMS sticking to the mold, the silicon oxide masters FIG. 1: (a, d) AFM images of SiO2 molds, (b, e) stiff 10:1
PDMS (1.6 MPa) and (c, f) soft 50:1 PDMS (17 kPa) replicas. The PDMS surfaces were scanned in 1% Triton X-100.

were silanizated in vacuum using 1H, 1H, 2H, 2Hperuorooctyl-trichlorosilane (Sigma, St. Louis, MO). Two types of mold patterns were used: the rst one consisted of a square lattice with 2.5 m squares and lattice constant of 3.5 m (Fig. 1a) and the second one had long trenches of 2, 5, 10, 20, 50, 70 m width, with a 100 m spacing in between (Fig. 1d). Here it will be demonstrate how the competition between surface free energy and elastic (bulk) energy will modify the surface prole of a solid. Usually, stamps for micro-contact printing, micro-uidic channels and micro-textured surfaces are produced using PDMS mixtures of 10:1 (base:to cross-linker) [2] giving accurate replicas at this length scale (micrometer). In order to illustrate the strong elasticity effect, PDMS rubber with Young moduli of 11, 17, 20, 48, 93, 144 kPa and 1.6 MPa was molded from mixtures of 55:1, 50:1, 40:1, 30:1, 35:1 and 10:1, respectively. The comparison between 10:1 and 50:1 can be seen in Figure 1. The shape change due to the surface tension and elasticity can be calculated starting from a sinusoidal surface prole with amplitude h0 and wavelength : u0 = h0 cos 2x .

We will assume that h0 << . In this case the re-

116

laxed" surface prole u1 would still be sinusoidal, with a reduced amplitude h1 , but the same wavelength. Thus, the surface displacement in normal direction is u = u0 u1 = (h0 h1 )cos 2x ,

corresponding to the local pressure distribution [3] p= E (h0 h1 ) cos 2x ,

where E = E/(1 2 ), with E the Young modulus and the Poisson ratio. Thus, the elastic energy can be written as Eel = 1 2 d2 x u(x)p(x) = A0 E (h0 h1 )2 , 4 FIG. 2: Measured (solid lines) and calculated (dashed
lines) height proles of lines molded in 11 kPa PDMS using 2-, 5-, 10-, and 20- m -wide lines molds of 531 nm depth. Theory curves were calculated using an interface tension of 8.5 mN/m.

where A0 is the nominal area of the surface. The surface energy is:
2

Es =

d x 1+

du1 (x) dx

1 2

A0

2 2 h1 . 2

Minimizing the total energy Eel + Es with respect to h1 gives h1 = h0 4 h0 1 1 + 4/E E . (1)

For surface proles characterized by some width and some height h0 we expect the scaling h = h0 h1 h0 E (2) FIG. 3: Normalized height change of the PDMS replicas
produced in lines molds (1d) with depths of 531, 299, 184, and 93 nm. Error bars denote the standard deviation of the values measured at the different mold depths. Experimental results for line widths of 2 m (triangles) and 5 m (squares) are shown along with the theoretical results, with an interface tension of 8.5 mN/m.

In general a surface with a variation in one direction can be mathematically described in the Fourier formalism as: h0 (x) = dqh0 (q)eiqx (3)

with q = 2/. Then the relaxed surface prole is h0 (x) = dq h0 (q)eiqx 1 + 2q/E (4) Even for stiff PDMS ( 2M P a) molding delity will become problematic at smaller length scales, equation (2) giving a good analytical expression to the rule of thumb the stiffer the better. Equation (4) can be used for exact surface topography calculation of the resulting replica molding samples when the elasticity and the surface tension of the material are known. An inverse approach would give a direct measure of the surface tension for soft solids in the case of known Young modulus and expected surface prole.

Starting with a surface prole like the one in Figure 1e for h0 (x), the surface prole from Figure 1f can be calculated using Equation (4). The calculated proles for the 2-, 5-, 10-, and 20- m -wide lines compared with the experimentally measured ones are presented in Figure 2. As the AFM scan was done in Triton X-100 a surface tension of 8.5 mN/m and a stiffness of 11 kPa were considered in the calculation. Equation (2) indicates that the expected change in height is proportional to mold depth. This is indeed visible in Figure 3 which shows the normalized height change for the 2 and 5 m lines produced in different molds. Moreover, h/h0 measured at the center of the line can be predicted well within the error bars of the AFM measurement knowing only the elasticity, Poisson ratio and the surface tension of the material.

[1] O.D. Gordan, B.N.J. Persson, C.M. Cesa, D. Mayer, B. Hoffmann, S. Dieluweit, R. Merkel - Langmuir 24, 6636 (2008) [2] Y. Xia, G. Whitesides, Annu. ReV. Mater. Sci. 28, 153 (1998) [3] H. Westergaard, Trans. Am. Soc. Mech. Eng., J. Appl. Mech. 6, 49 (1939)

117

Presenilin 1 Affects Focal Adhesions and Cell Forces via c-Src Regulation
D. Waschbsch1, S. Born1, V. Niediek1, N. Kirchgener1, I.Y. Tamboli2, J. Walter2, R. Merkel1, B. Hoffmann1
1 2

IBN-4: Biomechanics Department of Neurology, University of Bonn

Presenilin 1 (PS) is a critical component of the -secretase complex that cleaves transmembrane proteins. This process plays an essential role in signal transduction and vital functions such as cell adhesion. Here, we -/found for PS1 cells an altered morphology with significantly reduced sizes of focal adhesion sites (FAs) compared to wild-type. Furthermore, cell forces were reduced by 50%. PS1 deficiency was also associated with decreased tyrosine phosphorylation levels of FA specific proteins, which was caused by a transcriptional down-regulation of c-Src kinase. The direct regulatory connection between PS1 and c-Src is ephrinB2, a receptor mediating cell-cell adhesion. EphrinB2 becomes cleaved by PS1 with a subsequent translocation of the ephrinB2 intracellular domain (ICD) into the nucleus, acting as coactivator for c-Src transcription. Therefore, we conclude that -secretase is vital for controlling cell adhesion by transcriptional regulation of c-Src via ephrinB2 cleavage.

Cell analyses were performed using wild-type mouse embryonic fibroblasts as well as PS1-/mutant strains. Cell biological procedures were performed according to standard protocols. Cells were either analyzed using phase contrast or transfected with GFP-fusion proteins and analyzed by fluorescence microscopy. Alternatively, cells were fixed and proteins were stained by immunolabelling. Northern blot, western blot as well as qRT-PCR experiments were performed according to standard protocols. Elastomeric substrates were prepared and calibrated (Youngs modulus = 13 kPa, Poissons number = 0.5) as described earlier (3). To characterize the exact function of PS in cellmatrix adhesion processes wild-type (WT) and presenilin 1 knock out mouse embryonic fibroblasts were cultured and used for immunofluorescence analyses against actin and vinculin as marker of focal adhesion sites (Fig. 1). WT cells were characterized by well visible FAs. These sites with an average size of 0.9 m were mainly located at the cortex of cells and had an elongated shape. To every FA thick F-actin bundles, called stress fibers, were connected. -/Very different results were found for PS1 cells. Here, the number of FAs was reduced by 40%. The average size of FAs was diminished by 30 to 40% and reached 0.65 m on average. Their spatial distribution changed from a cortical to a disperse localization. Prominent actin stress fibers were absent. For a quantitative measurement of cell forces WT -/as well as the PS1 mutant strain were seeded on micropatterned, soft PDMS-substrates. Displacements of the regular micropattern were visualized by RICM and the generating forces were calculated from these data. For single FAs of WT cells, analyses revealed forces in the range of 13 nN (Fig 1B) with a generalized first moment of -/about 8.5 pNm (=4.7 pNm; n=86). PS1 cells instead applied forces in the range of just 7 nN per focal adhesion with a generalized first moment of 3.0 pNm (=3.1pNm; n=36). Since earlier experiments indicated an influence of PS on ephrinB1 putatively affecting c-Src activity, we analyzed the phospho-tyrosine levels of FAs in WT and PS1-/-.

PS1 is an aspartyl protease forming the active components of the -secretase complex, which cleaves transmembrane proteins within their transmembrane domains leading to the release of the two cleavage products from the membrane (1). The freed products can play important roles in signalling pathways by translocating into the nucleus and acting as transcriptional coactivators (2). Presenilin affects many mechanisms and one of these is the ephrinB/Eph receptor mediated cellcell adhesion. Since ephrins additionally bind to cellular sarcoma protein kinase (c-Src), a protein vitally involved in focal adhesion formation, it is speculated that PS might be an important regulator for switching cell function from a sessile to a dynamic, moving phenotype. Here, we identified a strong reduction in FA size upon PS1 deficiency associated with a reduction in cell force formation by over 50%. Both effects went along with a decreased phosphorylation level of FA associated proteins and were caused by a PS1 dependent downregulation of c-Src on the level of transcription via ephrinB2 cleavage.

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Fig. 2 Crude protein (A) as well as total RNA (B) were -/isolated from WT and PS1 and analyzed for indicated proteins/mRNAs. -tubulin and 28S rRNA, respectively, were used as internal standards

Fig. 1 (A) WT as well as PS1 cells were stained for actin and vinculin in immunolabelling experiments. (B) Cell types as in (A) were grown on elastomeric substrates and substrate deformations (yellow) were determined. Under-lying forces applied at every FA (red) as well as the generalized first moment (blue) were calculated as described in Cesa et al. (3).

-/-

Life cell imaging revealed intense nuclear localization of EB2ICD-GFP (Fig. 3A). FAs of transfected PS1-/- cells were also restored in tyrosine phosphorylation intensity and size (1.0 2 m, =0.4 m , n=100 FAs) (Fig. 3A). Furthermore, qRT-PCR experiments revealed a strong increase of c-Src mRNA levels in these -/cells compared to untransfected PS1 cells. A similar increase was observed for c-Src in western analyses (not shown). Although toxic at high concentrations or enhanced incubation times, a full length EB2-GFP construct, expressed in WT and PS1-/- cells, identified the dependency of EB2-ICD translocation on PS1 function. While nuclear localization could be observed for EB2-GFP in WT cells no such signal was present in PS1-/- (Fig. 3B). These data proof EB2 to be cleaved by -secretase and that an EB2 cleavage product is transduced into the nucleus were it functions as transcriptional coactivator for c-Src.

These analyses were performed in fixed cells using a phospho-tyrosine specific antibody and revealed high levels of phosphoryla-tion in FAs of WT cells while phosphorylation of PS1-/- FAs was almost absent (data not shown). Since phosphorylation in FAs is mainly performed by activated c-Src kinase we analyzed the activation status of this kinase at tyr418 as well as -/its expression level in WT and PS1 cells. As given in Figure 2A, c-Src phosphorylated at tyr418 was decreased by 90% in the absence of PS1 compared to WT. As such result might have been caused by either regulation of autophosphorylation or regulation of expression, northern as well as western analyses for c-Src were performed. Protein levels of c-Src were reduced by 60% in -/PS1 cells (Fig. 2A). Northern analyses identified an almost identical reduction of c-Src transcripts. Here, levels of c-Src mRNA were reduced by 50% -/in PS1 cells (Fig. 2B). Reduced levels of c-Src protein were therefore likely caused by transcriptional downregulation rather than increased protein degradation. EphrinB2 cytoplasmatic domain is translocated in a PS1 dependent manner into the nucleus. To complete the signal transduction pathway from PS1 function to c-Src activity, we checked secretase targets for a putative influence on c-Src. Since two of them, ephrinB1 and ephrinB2, were also known binding partners of c-Src, we analyzed ephrinB2 in more detail. The experiments revealed c-Src regulation mainly at the transcriptional level. We therefore tested ephrinB2 intracellular domain (EB2-ICD) for transcriptional co-activator function. An EB2ICD-GFP construct was expressed in WT as well as PS1-/- cells and its localization was compared to GFP only.

Fig. 3 (A) PS1 cells were transfected with an EB2ICDGFP construct and analyzed in fixed cells for GFP (green) as well as tyrosine phosphorylation of FAs. (B) -/WT and PS1 cells were transfected with full length EB2GFP and analyzed for nuclear translocation of an EB2GFP fragment. Note that translocation was only detected in the presence of functional PS1.

-/-

[1] B. De Strooper, W. Annaert, P. Cupers, P. Saftig, K. Craessaerts et al. (1999) Nature 398, 518-522 [2] A. Georgakopoulos, P. Marambaud, S. Efthimiopoulos, J Shioi, W. Cui et al. (1999) Mol Cell 4, 893-902 [3] C. M. Cea, N. Kirchgener, D. Mayer, U. S. Schwarz, B. Hoffmann, R. Merkel (2007) Rev. Sci. Instr. 78, 034301

119

Heart Muscle Cells Suspended Between Elastic Micropillars

A. Kajzar1,2, N. Hersch1, C.M. Cesa1, N. Kirchgener1, B. Hoffmann1, R. Merkel1
1 2

Institute of Bio- and Nanosystems 4: Biomechanics Marian Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, 30-059, Krakow, Poland

In living animals cells are parts of tissues where they experience a soft environment of polymers and neighbouring cells. However, classical cell culture employs hard and flat substrates such as polystyrene plates for cell attachment. These highly artificial culture conditions lead to cell adaptation which often severely compromises experimental results. Here we developed micropillar arrays from soft rubber as cell culture systems. Heart muscle cells were cultivated within these systems. A comparison of these cells with others that were grown on flat surfaces of identical material revealed much closer to nature cell morphology on micropillars. Moreover, spontaneous myocyte beating lead to micropillar bending that was exploited to determine cell forces. Cell force and micropillar stiffness showed a clear correlation indicating amplitude as decisive feed-back signal for the control of cellular contraction. Within micropillar arrays we cultivated rat heart muscle cells (cardiac myocytes). Cells strongly preferred to span between the elastic micropillars over adhesion to the underlying flat substrate. In addition, the architectures of the cytoskeleton and of protein complexes formed for adhesion were strongly dependent on the environment of the cell. On flat parts of the substrates we observed prominent stress fibres and focal adhesion sites. In contrast, cells suspended between micropillars exhibited well organized myofibres and costameric adhesions at the locations of Z-bands as found in heart tissue. These observations argue for close to nature environmental conditions within elastomeric micropillar arrays. Furthermore, these micropillars could be exploited to determine cell forces. To this end we derived closed expressions for the stiffness of elastically founded pillars. Sylgard 184 (PDMS*) was carefully mixed in a 20:1 ratio (base:cross-linker) and dispensed onto a microstructured SU-8 master. Subsequently, system was overlayed by a cover slide and PDMS* was crosslinked at 60C. The next day, cover slides with PDMS micropillars on top were peeled off the masters and glued to the bottom of perforated Petri dishes. Before use, micropillars were fluorescently labelled with DiD [1]. Myocytes were isolated from 19-day old Wistar rat fetal pups as described [2]. 30.000 cells were added to a micropillar substrate and analyzed 2 to

3 days after. Cells were either fluorescently labelled with calcein or fixed for immunofluorescence experiments. Analyses were performed on a confocal microscope (Zeiss, LSM510).

FIG. 1: Myocytes stained for -actinin (red). Left:

Myocyte suspended between micropillars. Image was taken at a height of 20 m above pillar base. Note that all sarcomer units are aligned in parallel forming the classical striated muscle morphology whereas this morphology is severely disturbed on flat substrates (right). Right: Mycocyte grown on flat substrate besides micropillars. Note the identical scales.

FIG. 2: Laser scanning microscopy images of pillars (red) with adhered cells (green). Side views. Pillar diameters, 10 m.

120

probe the impact of substrate geometry on these cells, we developed an array of elastomeric micropillars as cellular environment. Freshly isolated myocytes were seeded on these arrays and analyzed. Size of myocytes adhered to micropillars decreased compared to those adhered to flat substrate, cf. Fig. 1. In addition, cells were characterized by a strongly reduced number of actin stress fibres. Furthermore, cells grown within micropillar arrays avoided adhesion to the elastic base of the micropillars. Instead, myocytes exclusively span between the pillars for pillar distances of 10 to 30 m (Fig. 2). In order to characterize the force generation system of myocytes grown between elastic micropillars in more detail, we determined cell forces from micropillar bending, cf. Fig. 3. Despite the fact that pillar bending is a classical application of continuum mechanics, no closed expressions for the stiffness of micropillars emerging from a substrate of identical material were available. Therefore we derived an approximate treatment based on the Boussinesq theory of the deformation of an elastic half space under the influence of mechanical forces. This approach revealed that micropillars emerging from identical material are by about 30% softer compared to micropillars with clamped bases, i.e. the commonly made assumption overestimates cell forces substantially. Evaluating cell forces we found a clear cut correlation between micropillar stiffness and cell forces. This can be easiest explained by cell contractions being regulated to achieve a specific amplitude, a conclusion which was further supported by the fact that the slopes of the regression lines coincided with the mean cell contractions in relaxed and contracted state. Taken together our data clearly show that close to nature cell culture can be achieved using elastic micropillars. As a further benefit, these microsystems enable reliable cell force measurements and valuable insight into mechanical regulation of cellular processes. Applications in functional drug studies on the single cell level are foreseen.

FIG. 3: Myocytes were grown within labelled micropillar arrays for two days. Using a confocal microscope pillars were imaged at a height of 15 m above the base. A: Pillar positions during cell relaxation (top), and contraction (bottom). B: Micropillar displacements along (full lines) and perpendicular (broken lines) to cell orientation. C: Forces of many cells during contraction (filled boxes) and in the relaxed state (open boxes). The lines are linear regressions. Full line: contraction forces, slope 1.6 m; broken line: forces in the relaxed state, slope 0.6 m. Myocytes are a highly organized cell type in heart tissues. They are characterized by repetitive units of actin-myosin fibres, called sarcomers. These units fill the whole cell with parallel orientation to each other and are connected to the environment by specific adhesion structures, named costamers [3]. In contrast, neither the high organization of sarcomers nor costameric adhesion structures can be found when myocytes are cultured on flat and stiff substrates as e.g. classical culture dishes. To

[1]

Kajzar, A., C.M. Cesa, N. Kirchgener, B. Hoffmann and R. Merkel, Biophys. J. PMID: 17981895 (2007)

[2] Cesa, C.M., N. Kirchgessner, D. Mayer, U.S. Schwarz, B. Hoffmann and R. Merkel, Rev. Sci. Instrum. 78:034301 (2007) [3] Alberts, B., D. Bray and J. Lewis, Molecular Biology of the Cell, W.H.Freeman & Co Ltd, Oxford (2006)

121

Diffusion and Membrane Adhesion: Avidin and E-Cadherin Case Study

S. Fenz , K. Sengupta
1 1 2 1, 2

, R. Merkel

IBN-4: Biomechanics Centre Interdisciplinaire de Nanoscience de Marseille, CINAM/CNRS-UPR3118, France

Cell-cell adhesion is a highly complex process of vital importance for any multicellular organism. An abundance of proteins and signal cascades contribute. Nevertheless, in early stages physical forces dominate as the cell needs some time to initiate an active response. Here, we present a biomimetic model system for cell-cell adhesion mediated by mobile receptor-ligand pairs. We study quantitatively the role of the membrane uidity and the diffusivity of membrane-bound receptors in biomembrane adhesion applying bleaching techniques. Membrane adhesion is characterized with respect to the inter-membrane distance by high precision micro-interferometry. In recent years, structure and dynamics of the essentially uid cell membrane and its nanoscale inhomogeneities have attracted much interest. Diffusion of lipids and proteins in the plane of the membrane impacts directly on the local dynamic structure of the membrane. Diffusion thus plays a vital role in many membrane related functions including adhesion, recognition and transport. To quantitatively investigate the impact of individual parameters on the uidity and structure of the membrane, well-dened model systems were developed. The scope of this project was to elucidate the underlying physical principles. We applied Continuous Photobleaching (CP) and Fluorescence Recovery after Photobleaching (FRAP) to study membrane uidity as a function of specic protein binding to ligands within the membrane and depending on binding of a second membrane. Membrane adhesion was effected either by biotinneutravidin (an avidin analogue) or the extracellular domains of the homophilic cell adhesion molecule E-cadherin (see FIG 1). To determine the resulting inter-membrane distance with high precision ( 4 nm), traditional Reection Interference Contrast Microscopy (RICM) theory was extended to account for the reections from all ve relevant interfaces. For neutravidin the fate of the receptors during membrane adhesion was additionally monitored. A solid supported lipid bilayer (SLB) served as a model for the rst membrane. It was prepared by the LangmuirBlodgett Langmuir-Schfer technique and contained a controlled percentage of ligand lipids. We used biotinylated lipids to bind neutravidin and NTA lipids

to bind the E-cadherin Fc chimera via its histag (for details on the chimera see FIG 3). Giant unilamellar vesicles, prepared by electro-swelling, were employed to mimic the second membrane.

Sketches of the model systems illustrating the membranes and binding molecules involved. Left: biotinneutravidin, right: E-cadherin-E-cadherin.
FIG. 1:

We found that in case of strong receptor-ligand interaction (biotin-avidin, Gibbs free energy of bonding G0 in solution 35 kB T) binding of soluble receptors to the SLB alone led to reduced diffusion of tracer lipids reecting a decrease in the overall membrane uidity [1]. This effect scaled with the concentration of receptors (see Table 1). From theoretical considerations, the decrease could be attributed partially to introduction of obstacles [2] and partially to viscous effects [3]. The obstacles were formed by biotinylated lipids pairwise connected to the same neutravidin molecule. This formed one large object, which diffused more slowly than single lipids or was completely pinned depending on the receptor concentration. Moreover, the viscous protein layer on the distal side of the SLB introduced extra friction.

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biotin bare +NAV +NAV+GUV 1% 2.30.2 2.10.2 1.80.2 5% 2.20.2 1.70.2 1.50.2
Diffusion constants [m2 /s] of the tracer lipid in the SLB before and after binding of protein or vesicle for various concentrations of biotinylated lipids in the SLB. Error bars represent single standard deviations.
TAB. 1:

Specic binding of a GUV membrane led to additional slowing down of tracers (up to 15%) caused by an increase in obstacle density and enhanced friction due to the adhering membrane. Receptors were accumulated in the adhesion zone till full coverage corresponding to 5% biotinylated lipids was achieved (see FIG 2). Bleaching experiments on the receptors themselves revealed a fast decrease in receptor mobility with increasing receptor concentration. At full coverage the receptors were practically immobilized [1].

The exact E-cadherin binding conguration is still under discussion. So far, three different scenarios were proposed (see FIG 3) corresponding to hth,1 = 52 nm, hth,2 = 44 nm and hth,3 = 36 nm. We measured hexp = (54 9) nm strongly favoring the scenario with only the outmost domain involved [1]. As a result, the inter-membrane distance in the E-cadherinE-cadherin case was signicantly larger than in the biotin-avidin case. Consequently, the adhering membrane generated less friction.

3: Three different binding congurations of Ecadherin-E-cadherin are under discussion: overlap of the outmost domain, the three outmost domains or all domains. FIG.

In summary, we gained three new insights:

a) The impact of protein binding and protein mediated membrane binding on the uidity of a membrane depends strongly on the geometry of the protein - especially on the number of binding sites and their distance as well as the closeness of the interaction. b) The high precision micro-interferometric measurements of the inter-membrane distance allowed to shed light on the hitherto unclear binding conguration of the cell adhesion molecule E-cadherin. We conclude that E-cadherin molecules residing on a soft, exible membranes bind predominately with their rst domain. c) The adhesion assays in a cell-free model system unequivocally show that accumulation of mobile receptors does not require active interaction of a cell. These results on diffusion, inter-membrane distance and receptor accumulation should contribute to the understanding of equivalent phenomena in cell-cell adhesion.

(a) RICM micrograph of an adhering vesicle. (b) Reconstructed height [nm] in the adhesion disc. (c) Fluorescence micrograph of accumulated neutravidin in the adhesion disc. The initial biotin concentration on the SLB was 2%. (d) Intensity prole along the white line in c. The scalebar is valid for a-c: 10 m.
FIG. 2:

In case of weak receptor-ligand interaction (Ecadherin-E-cadherin, G0 2 kB T) no signicant change in diffusion of tracer lipids was observed upon protein binding and subsequent vesicle binding [1]. At rst glance, the lack of retardation after receptor binding is surprising, since the Ecad construct is a dimer and its binding should therefore have introduced, as in the strong binding case, mobile obstacles in the form of coupled NTA lipids. Nevertheless, the size of the weak protein-lipid complex was only one third of the strong one. Thus, it had no measurable effect on the tracer lipid diffusivity. In order to understand the lack of retardation after vesicle binding the inter-membrane distance for both cases was analyzed. We measured hexp = (7 1) nm for biotinneutravidin. This height was in very good agreement with the protein and linker dimensions known from crystallography and X-ray reectivity (hth = 5.6 nm).

[1] S. F. Fenz, R. Merkel and K. Sengupta, Langmuir 25(2), 1074-1085 (2009). [2] M. J. Saxton, Biophysical Journal 52(6), 989-997 (1987). [3] E. Evans and E. Sackmann, J. Fluid. Mech. 194, 553561 (1988).

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New Role for Filopodia in Adhesion Formation During Cell Migration

C. Schfer, B. Borm, S. Born, C. Mhl, E.M. Eibl, B. Hoffmann
IBN-4: Biomechanics
Cell migration is a decisive prerequisite for embryogenesis, wound healing and immune defence. The exact interconnection between focal adhesion (FA) formation and correct guidance is the basis for an efficient functioning of these major biological processes. FAs connect the cytoskeleton of the cell to the environmental matrix to provide anchorage and force transmission. We found an essential role for filopodia in the formation of FAs during cell migration. Since now, FAs were thought to be built up in the lamellipodium. Here we show that nearly all FAs depend on stable filopodia. These FAs were formed along the axis of filopodia containing all tested adhesion proteins and were just increased in size when reached by the lamellipodium. Blocking filopodia fully inhibits FA formation. Therefore, filopodia are the key factors for formation and localization of all FAs and thus for accurate cell movement and direction. Keratinocytes are epithelial cells derived from human skin which effectively migrate after stimulation. While migrating they form a wide lamellipodium with long finger-like structures, so called filopodia, in direction of migration (see FIG. 1 A). The lamellipodium as characteristic feature at the leading edge of motile cells creates the necessary force to pull the front of the cell in direction of migration via actin polymerisation [1] and locates the origins of new focal adhesions [2]. In contrast, filopodia are thin membrane extensions comprising tight bundles of parallel actin filaments with their proximal end embedded in the lamellipodial actin network. Functionally, filopodia are described as structures mainly probing the substrate for proper matrix composition but how filopodia integrate the substrate information into lamellipodial processes especially formation of new adhesion sites is still unclear although association of adhesions with filopodia has been described [3].

FIG. 2: Protein content of filopodial extensions. Motility induced keratinocytes were fixed and immunofluorescently labeled using antibodies against focal adhesion site specific proteins paxillin (A), talin (B) and vinculin (C). Localization of GFPtensin (D), GFP VASP (E) and GFPzyxin (F) were analyzed in living cells. Here, keratinocytes were transfected one day before EGF stimulation and subsequently analyzed by confocal microscopy. White arrows indicate fluorescent signals within filopodia in fluorescence and phase contrast. Note that all proteins can be found in the filopodia extensions. Scale bar=10 m.

FIG. 1: Correlation between filopodia and focal adhesion sites. Keratinocytes were grown for one day and subsequently motility induced by EGF (epidermal growth factor). Confocal images in phase contrast (A) and in reflection (B) are shown. Note the localization of focal adhesion sites right behind stably adhered filopodia (black arrows in A and B). Direction of keratinocyte movement=white arrow. Scale bar=10 m.

Many motile cells such as keratinocytes exhibit a highly dynamic formation and disassembly of adhesion sites. These adhesion sites become prominent at the leading edge of lamellipodia with an elongated shape upon movement. In our experiments we focus on the interrelation between filopodial and lamellipodial adhesion structures. Strikingly, lamellipodial adhesions were always located in direct extension of filopodia (see FIG. 1). Lamellipodial adhesions were always found right behind stable filopodia whereas cell areas with filopodia unable to attach were characterize by the absence of lamellipodial adhesions. The given data strongly argued for an important function of filopodia in adhesion site formation of moving cells. To analyze if adhesion structures can already be found in filopodia the protein localization of focal adhesion site specific proteins

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like paxillin, talin, tensin, the vasodilator-stimulated phosphoprotein (VASP) and vinculin was tested by immunofluorescence as well as in living cells using green fluorescent protein (GFP)-fusion constructs (see FIG. 2). These data clearly show that each of these proteins is localized in filopodia most likely along their complete lengths with signal intensities highly above cytoplasmic background. To our surprise the same localization was also found for zyxin, a marker protein for mature focal adhesions (see FIG. 2 F). These data suggest that stable filopodia form small but fully assembled focal complex like structures termed here filopodial focal complexes (FX).

Cells transfected with GFPvinculin identified a full correlation between the neomycin induced migratory block and inhibited focal adhesion formation. Furthermore, FRAP (Fluorescence Recovery After Photobleaching) analyses on these cells revealed a full maturation state of focal adhesions formed before neomycin treatment but still located at the leading edge of polarized cells (see FIG. 4 B). These results were highly comparable to those found in [4] for focal adhesions of sessile, neomycin untreated keratinocytes while nascent adhesions of migrating cells contained a much higher fraction of exchanging vinculin (see FIG. 4 B, red curve).

FIG. 3: Growth of filopodial FXs to focal adhesions. Keratinocytes were transfected with tensin as GFPfusion protein and motility stimulated after 24 h of growth. Stably adhered filopodia were analyzed over time in phase contrast (top) and fluorescence (bottom). White arrows indicate filopodial FXs at time point 0 changing to focal adhesions over time. Same position is also indicated in phase contrast in the first image of the series. Note the strong increase in fluorescence as soon as the lamellipodium reaches the filopodial adhesion. Time points are given in seconds. Scale bar=3 m.

FIG. 4: Blocking filopodia inhibits motility and adhesion formation: (A) Migration speeds of EGF stimulated keratinocytes with (n=74) and without (n=62) neomycin treatment were analyzed and are indicated as boxplots. Migration speeds are significantly different (pvalue<0.001). (B) GFPvinculin transfected cells were treated with neomycin. Afterwards, focal adhesions at the leading edge were analyzed by FRAP. Mean value and standard deviation for every time point from normalized curves are given (blue, n=13). For better visualization data were overlaid with experiments on focal adhesions at the leading edge of migrating, neomycin untreated cells (red, n=14, [4]). A single exponential model was fitted to the data describing a simple binding-unbinding kinetics.

Taken all these data together we present new insights in formation and function of filopodia in migrating keratinocytes. It becomes clear that filopodia play a fundamental role during adhesion site formation and thus determine the localization and shape of almost every adhesion site.

We clearly showed that adhesion structures can already be found in filopodia. Therefore we analyzed adhesion formation in filopodia using live cell imaging (see FIG. 3). Here, all tested adhesion proteins were present along stably adhered filopodia. Their localization labeled parts of the filopodia or covered their whole lengths but was always elongated. As soon as the filopodial adhesion proteins were reached by the lamellipodium, the size of the adhesion structure increased along the former orientation of filopodia. During this enlargement process the spatial orientation of the focal adhesion did not change. These data illuminate that form and localization of focal adhesions is determined by filopodial adhesions. Since our data on moving keratinocytes clearly showed a dependency of focal adhesion sites on filopodial adhesions, we analyzed whether focal adhesions would be formed in the absence of filopodia. For this reason we blocked filopodia formation by neomycin supplementation. In the presence of neomycin all cells (n=338) were significantly reduced in migration (see FIG. 4 A).

[1]

A. Ponti, M. Machacek, S.L. Gupton, C.M. Waterman Storer, G. Danuser, Two distinct actin networks drive the protrusion of migrating cells, Science 305 (2004) 17821786. R. Zaidel-Bar, C. Ballestrem, Z. Kam, B. Geiger, Early molecular events in the assembly of matrix adhesions at the leading edge of migrating cells, J. Cell Sci. 116 (2003) 46054613. M. Nemethova, S. Auinger, J.V. Small, Building the actin cytoskeleton: filopodia contribute to the construction of contractile bundles in the lamella, J. Cell Biol. 180 (2008) 12331244 C. Mhl, N. Kirchgener, C. Schfer, K. Kpper, G. Diez, W.H. Goldmann, R. Merkel, B. Hoffmann, Vinculin exchange dynamics regulates adhesion site maturation and adhesion strength, unpublished.

[2]

[3]

[4]

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The Mechanism of Adhesion Strength Adaptation in Living Cells

C. Mhl1, N. Kirchgener1, C. Schfer1, K. Kpper1, G. Diez2, W.H. Goldmann2, R. Merkel1, B. Hoffmann1
1 2

IBN-4: Biomechanics Center for Medical Physics and Biology, Friedrich-Alexander-University of Erlangen-Nuernberg maturation state, the phosphorylation of FAs was significantly lowered (see FIG. 1).

The morphogenesis and architecture of multicellular organisms is determined by the coordinated adhesion of single cells to each other and to the extracellular matrix (ECM). The mechanical connection between the actin cytoskeleton and the ECM is provided by protein clusters at the cell membrane building distinct adhesion spots which are denoted as focal adhesion sites (FAs). Many proteins incorporated in FAs are constantly exchanging within a timescale of seconds. Here we show, that the exchange of the FA-protein vinculin decreases with proceeding age of FAs and is regulated by phosphorylation at a single tyrosine residue of vinculin. This stabilization of vinculin within the FA goes along with an enhanced force transduction ability of the cell. Based on these findings we consider the phosphorylation-regulated incorporation of vinculin as a mechanism to modulate adhesion strength. We therefore propose a new role of vinculin as a tuneable factor for the mechanical stability of FAs. Migrating cells are excellent model systems to study the controlled assembly and disassembly of FAs, since their movement is based on a constant adhesion turnover with formation of new adhesions at the front and the release of old adhesions at the cells rear. During their lifetime, FAs undergo a regulated development from a nascent to a mature state by changing size, shape and protein composition. Nascent FAs are formed at the front of the migrating cell. During their maturation they increase in size remaining relatively stable in respect to the substrate. While the cell-body moves forward they come closer to the trailing edge of the cell where they finally dissolve. Thus, the coordinated formation and release of FAs determines the direction of migration and is thought to play a key role in directed cell movement like chemotaxis [1]. We investigated the phosphorylation and exchange dynamics of the FA protein vinculin in epithelial cells from human skin (keratinocytes) migrating on a flat surface. As an adaptor protein, vinculin connects other FA proteins with actin filaments and is thought to stabilize the adhesion complex [2]. Quantitative immunostaining studies revealed a high vinculin phosphorylation in the nascent FAs at the cell front. With increasing

FIG. 1: Phosphorylation of vinculin in focal adhesions (FAs) of migrating skin cells. A: Pseudocolor image of the ratio between phosphorylated vinculin and total vinculin within FAs of a migrating cell. The cell shape is marked by a white line and the big arrow points to the direction of migration. Nascent FAs at the cell front (arrowheads) are higher phosphorylated than mature FAs located behind. B: Amount of vinculin phosphorylation in FAs in respect to their distance from the leading edge. Asterisks indicate significant differences of vinculin phosphorylation compared to FAs of stationary cells. Error bars indicate standard deviation. Data were collected from 38 cells.

Since phosphorylation events are known as a mechanism to regulate the binding ability of certain proteins, we explored the exchange kinetics of vinculin in respect to the FA maturation state. Therefore, migrating keratinocytes were transfected with a gene for fluorescently labelled vinculin making it possible to visualize this protein in vivo by light microscopy. To measure the vinculin exchange in single FAs, we photobleached vinculin at these sites and measured the fluorescence recovery over time. If there was no protein exchange, the fluorescence wouldnt recover at all. In contrast, if all bleached proteins were replaced, the fluorescence would recover completely. Hence, with this method called Fluorescence Recovery after Photobleaching (FRAP) the amount of the stably incorporated against the constantly exchanging vinculin could be determined in single FAs (see FIG. 2 A).

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FIG.3: Force application during cell locomotion: Fluorescent image of vinculin in a migrating cell with FAs appearing as bright spots. Red arrows mark the force vector for each FA. The white arrow points to the direction of migration. Eigenvectors of the generalized first moment tensor are indicated by green arrows Mature FAs at the rear produce strong inwards directed forces. Nascent FAs at the front produce weak rearwards directed forces. Scale bar: 10 m. FIG.2: Vinculin exchange dynamics examined by Fluorescence Recovery after Photobleaching (FRAP). A: FRAP experiment at a mature FA of a cell transfected with fluorescently labelled vinculin. FAs appear as bright spots. The bleach-field is marked by a red circle. Bleaching occurs at 0s. Since vinculin is constantly exchanging within the FA, the fluorescence recovers over time. Scale bar: 5 m. B: Mean fluorescence recovery of vinculin over time from several FRAP experiments: nascent FAs at the cell front (red, n=14), mature FAs at the rear (green, n=11), FAs from sessile cells (blue, n=14), nascent FAs with phosphorylation-inhibited vinculin (grey, n=13). C: Saturation values of the mean recovery curves shown in B. This value indicates the exchanging fraction of vinculin. Asterisks designate a statistically significant difference from frontal (red) or rear (green) adhesions.

In these experiments, tractions of the migrating cell produced local substrate deformations which could be quantified by tracking the displacement of fluorescent beads embedded in the substrate. Since the elastic properties of the silicone substrate and the location of FAs as sites of force transduction were known, traction forces could be calculated from these deformations. These experiments revealed that major traction forces were transmitted at the cells rear where typically mature FAs are located (see FIG. 3). Taken together, these experiments confirm the direct interplay between the amount of stablely incorporated vinculin into the FA and its ability to transduce force, whereas the vinculin incorporation could be modified by phosphorylation. Thus, we suggest vinculin to be an important stabilization factor for FAs which is directly regulated by phosphorylation through intracellular signalling pathways.

Just like the amount of phosphorylation, the fraction of exchanging vinculin decreased with the FA maturation level indicating that vinculin exchange is regulated by phosphorylation. To verify this theory, cells were transfected with a slightly altered vinculin gene, where phosphorylation at a specific tyrosine residue (TYR1065) was inhibited. The exchange of the inhibited vinculin mutant was in nascent FAs significantly lowered compared to the wild type vinculin confirming the direct interplay between the phosphorylation of vinculin and its binding kinetics (see FIG. 2 B and C). As Vinculin is essential for mechano-coupling, the regulation of vinculin binding could be a mechanism to adapt the adhesion strength of individual FAs. Therefore, the correlation between FA maturation state and force transduction was examined by traction force microscopy of cells migrating on elastic silicone substrates [3].

[1] Webb DJ, Parsons JT, Horwitz AF, Nat Cell Biol 4(4):E97-100 (2002) [2] Ziegler WH, Liddington RC, Critchley DR, Trends Cell Biol 16(9):453-60 (2006) [3] Cesa CM, Kirchgessner N, Mayer D, Schwarz US, Hoffmann B, Merkel R, Rev Sci Instrum 78(3):034301 (2007)

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128

Education & Dissemination

International Helmholtz Research School International Soft Matter Conference 2007 IFF Spring School 2008 Jlich Soft Matter Days

129

International Helmholtz Research School of Biophysics and Soft Matter

The International Helmholtz Research School of Biophysics and Soft Matter (IHRS BioSoft) provides intensive training in biophysics and soft matter. It also offers a comprehensive framework of experimental and theoretical techniques that will enable PhD students to gain a deeper understanding of the structure, dynamics, and function of complex systems. In recent years, life science research has undergone a fundamental transition. It has become evident that even the simplest molecular machines display an astounding complexity, leaving alone networks of genes and proteins in a living cell. Thus, there is an urgent need for a more quantitative, theory-oriented approach. Soft matter research has, in parallel, made great progress in understanding the structure of complex multi-component macromolecular systems, their non-equilibrium behaviour and their response to external elds. A particular focus is laid upon unraveling the physics of biologically relevant systems. Thus, there is an urgent need for an interdisciplinary graduate education.

ular Sensory Systems headed by Prof. U. B. Kaupp in Bonn, the ICG-3 (Phytosphere) headed by Prof. U. Schurr in Jlich, the group on Molecular Physical Chemistry headed by Prof. C. Seidel in Dsseldorf, and the physical chemistry group headed by Prof. Strey in Cologne participate in the IHRS BioSoft. The research school accepts fellows for three-year PhD projects and is open to highly qualied and motivated applicants from all countries. The fellow PhD students will be based in one of the groups that are part of the IHRS, but also participate in interdisciplinary courses. Other students are welcome to join most of these courses as long as there are free places. The lectures of the school usually attract a number of extra participants that choose the topics selectively according to their needs. In 2007, two-semester introductory lecture courses, Introduction to Statistical Physics taught by Prof. Dhont and Prof. Gompper and Molecules of Life Introduction to the Chemistry and Biology of Cells with various lecturers from within the IHRS BioSoft (Prof. Schurr, Prof. Merkel, Prof. Kaupp, Prof. Seidel, Prof. Richter, Prof. Bldt, Prof. Willbold, Dr. Enderlein, and Prof. Offenhusser) were offered to the students. Both courses equip the students for their research projects with important basic knowledge: from a physics point of view, entropy and statistical physics have a large inuence on the behaviour of the systems that are usually mesoscopic. The biological lectures covered systems from amino acids to the structure and dynamics of entire cells as well as methods such as X-ray crystallography, uorescence spectroscopy, electrophysiology, and optical microscopy. In 2007 and 2008, the students learned about the important tool of Computer Simulations in Physics and Biology by a two-semester advanced-seminar course that covered various, independent talks on different topics: Monte Carlo and Molecular Dynamics Simulations, Polyelectrolytes, Solid State NMR, Evolution of Bacterial Genomic Networks, Mesoscopic Hydrodynamics, Colloids, Proteins, ProteinLigand Binding, Protein Structure Prediction, and Membrane Proteins. Most of the speakers were from Forschungszentrum Jlich and daily work with the methods and systems they presented: G. A. Vliegenthart, R. G. Winkler, H. Heise, M. Stoldt, M. Lercher, M. Ripoll, G. Naegele, A. Baumgrtner, M. Zacharias

The IHRS BioSoft is located at Forschungszentrum Jlich, run in cooperation with the universities in Cologne and Dsseldorf and caesar Bonn, and funded by the Helmholtz Association. Its ultimate goal is to advance the integration and exchange between physics, chemistry, and biology in research and education. Students benet not only from lectures, seminars, and lab courses given by experts in the eld, but also from courses in transferable skills. Furthermore, they experience the environment provided by a large, multidisciplinary research centre. In addition to groups within the PoF-BioSoft program, also the group Physics of Soft Matter headed by Prof. S. Egelhaaf in Dsseldorf, the group Molec-

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(IU Bremen), J. Granzin, and W. B. Fischer (NYMU, Taiwan). Very recently, students were offered the onesemester introductory lecture course on Cell Biology by Prof. Mller and Prof. Baumann that included lab demonstrations, and the advanced lecture courses on Complex Fluids by Prof. Strey (Cologne) and Rheology by Prof. Vermant (Leuven).

NMR as a tool to study protein structures (M. Schwarten, Structural Biochemistry) Non-genomic action of progesterone in human sperm (N. Goodwin, Molecular Sensory Systems, Bonn) Nanostructured gold electrodes for the functional coupling with neuronal cells (D. Brggemann, Bioelectronics) Microinterferometry: a tool to study membrane uctuations (C. Monzel, Biomechanics) Swarm behaviour of self-propelled particles (Y. Yang, Theory of Soft Matter and Biophysics) Photo-control of cell networks for extracellular recording systems (V. Maybeck, Bioelectronics)

Complementary laboratory courses provide the students with practical experience and strengthen the interdisciplinary approach. Every year, the two-week Neutron Scattering course (organized by T. Brckel, G. Heger, D. Richter, and R. Zorn) is open for the participation of IHRS students. The course provides an extensive training by theoretical lectures and practical exercises. In a course on Optical Spectroscopy, G. Schuetz (Linz), J. Enderlein (Tbingen), J. Humplickova (Prague), M. Sauer (Bielefeld), J. Hofkens (Leuven), T. Gensch, and J. Heberle (Bielefeld) taught several optical techniques, such as uorescence techniques, single-molecule spectroscopy, reaction-induced infrared difference spectroscopy, and Raman spectroscopy of biomolecules. The laboratory course Recording of Cell Activity for example on Ca2+ imaging in living cells was offered jointly by several institutes within the IHRS. In the last semester, a two-week course Fluorescence Spectroscopy by Prof. Seidel in Dsseldorf was open for IHRS fellows, a one-week course Cryo Transmission Electron Microscopy was organized exclusively for IHRS students by L. Belkoura and M. Baciu in Cologne and a one-day course on NMR Spectroscopy was offered by B. Knig in Jlich. The PhD students regularly present their research in the Students Seminar that is chaired by two IHRS BioSoft faculty members; every talk is followed by a long discussion. As the research in the participating groups, also the research topics of the PhD projects cover a wide range within biophysics and soft matter. Therefore a talk in the Students Seminar is very challenging, because it needs to be prepared such that physicists, chemists, and biologists can benet. Apart from questions and feedback about the research, the speakers usually receive also comments regarding the style of the presentation and whether it was suitable for the different parts of the audience. Topics of talks that were given include: Holographically induced nucleation (R. Hanes, Physics of Soft Matter, Dsseldorf) Regulation of HCN channels by phosphorylation (F. Winkhaus, Molecular Sensory Systems, Bonn)

Self-assembly in a binary H2O-C12E4 system (I. Savic, Physical Chemistry, Cologne) Squeezing actin: a TIRF microscopy study (A. Tsigkri, Soft Condensed Matter) Microemulsions as delivery systems (Sabine Schetzberg, Physical Chemistry, Cologne) Polyelectrolyte electrophoresis (S. Frank, Theory of Soft Matter and Biophysics) Molecular Dynamics simulations of polyethylene oxide (PEO) and PEO/PMMA blends (M. Brodeck, Neutron Scattering) Combined single-molecule force and uorescence spectroscopy (S. Grabowski, Molecular Physical Chemistry, Dsseldorf) How is shoot growth affected by low root temperature? (R. Poire, Phytosphere) HCN channels in the main olfactory bulb (A. Aho, Molecular Sensory Systems, Bonn) Morphologic and physiologic aspects of synaptic transmission in rat barrel cortex (G. Haack, Cellular Neurobiology)

Fellow PhD students already participated in two of the three seminars in transferable skills by Imperial College London that are organized by the Helmholtz Association. The seminars shall cover various aspects ranging from group work in the beginning of the thesis, presentation techniques up to writing of applications towards the end of the PhD project. Currently, rst students who have started their PhD projects within the IHRS BioSoft nish their thesis.

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International Soft Matter Conference, Aachen 2007

G. Gompper 1 , J.K.G. Dhont 2 , D. Richter 3
1 2 3

IFF-2: Theoretical Soft-Matter and Biophysics IFF-7: Soft Condensed Matter IFF-5: Neutron Scattering

The International Soft Matter Conference took place in Aachen on October 1-4, 2007. It brought together about 600 scientists from 35 countries to discuss all aspects of soft matter science.

With the large number of conferences organized every year on topics like polymer chemistry and polymer physics, colloid chemistry and colloid physics, surfactants in solutions, etc., the aim of an International Soft Matter Conference clearly had to be to bring together a balanced mixture of scientists working on all kinds of soft materials such as polymers, colloids, surfactants, membranes, biomaterials and their composites [2, 1]. The need for a unied view of Soft Matter systems is threefold. First, it has been recognized over the last decades that colloids, polymers and surfactants are by far not as distinct materials as previously assumed. Indeed, there is essentially a continuum of molecules and systems, which lls the triangle of materials illustrated in Fig. 1. The two main axes of this triangle are, roughly speaking, amphiphilicity as abscissa and elongation or exibility as ordinate. Let us illustrate this by following the left-hand side of the triangle from colloids to exible polymers. Traditionally, colloids are hard, spherical particles. However, there are also rod-like colloids. As the aspect ratio, the ratio of rod length and rod diameter, becomes larger, rods typically become more exible. An example is the fd-virus shown in Fig. 1. For even larger aspect ratios, the length exceeds the persistence length; this is the regime of semi-exible polymers, for which DNA is an example of enormous importance. Finally, in the limit of very small persistence lengths, we arrive at the classical, exible synthetic polymers. Second, mixtures of several components of colloidal, polymeric or amphiphilic character are becoming increasingly important, because they open up the possibility to tune and control material properties. Well-known examples are the depletion interaction between colloidal particles induced by polymers in solution, the intriguing mesophases in mixtures of spherical and rod-like colloids, the tuning of membrane properties by anchored polymers and amphiphilic block copolymers, or the modication of the properties of polymers melts by addition of colloidal particles to form nano-composites. Third, biological and biomimetic systems share many macromolecules and properties with Soft Matter sys-

FIG. 1: The components of modern Soft Matter systems

can be arranged in a triangle, which shown that there is a continuum of molecules and materials which lls the space between spherical colloids, exible polymers, and surfactants.

tems. Indeed, the application of physical concepts and ideas to biological systems has become one of the most intense activities in soft condensed matter in recent years. With more than 600 participants from about 35 countries (see Fig. 2), 8 plenary talks, 42 invited talks, 85 contributed talks, and about 400 posters, the amount of information provided during the conference was much too large to be summarized in a few lines here. Instead, we hope that the list of the plenary speakers and the titles of their talks will give a feeling about the exciting atmosphere and the intensity of the discussions during the conference: M. Cates (University of Edinburgh, UK) Lattice Boltzmann simulations of nonequilibrium complex uids. W. Gelbart (University of California, Los Angeles, USA) Physical aspects of viral infectivity. L. Leibler (ESPCI, Paris, France) Supramolecular plastics and rubbers. G. Maret (Universitt Konstanz, Germany) Elasticity, phonons and melting of colloidal crystals.

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FIG. 2:

Number of participants from countries with the largest participation.

D. Nelson (Harvard University, USA) Neutral mutations and gene surng in microorganisms. D. Roux (Saint-Gobain, Courbevoie, France) Glass technologies: colloids and active surfaces. H. Tanaka (University of Tokyo, Japan) Mechanical instability in phase separation, fracture, and cavitation. D. Weitz (Harvard University, USA) Dripping, jetting, drops and wetting: Structuring new soft materials with microuidics. The conference announcement is shown in Fig. 3. All invited speakers were invited to submit an original paper related to the subject of their presentation for a special issue of the European Physical Journal E [3]. All contributions were fully reviewed. This issue provides a good overview about the current status and topics of Soft Matter research. Due to the large interest and overwhelming participation in the conference, the program committee came to the conclusion that it would be a good idea to organize international soft matter conference every three years in the future. Participants were therefore asked for proposals. These proposals were presented in a plenary session. A public vote showed the largest support for the proposal from Granada (Spain). It will therefore the location of the International Soft Matter Conference 2010. We are looking forward to this meeting of the Soft Matter community.

FIG. 3:

Announcement of the International Soft Matter Conference 2007.

[1] European Network of Excellence Newsletter No.5, December 2007.

SoftComp",

[2] G. Gompper, J.K.G. Dhont, and D. Richter, Eur. Phys. J. E 26, 1 (2008). [3] Special Issue Eur. Phys. J. E 26 (2008).

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39th IFF Spring School: Soft Matter From Synthetic to Biological Materials

The 39th international IFF Spring School took place from 3 March until 14 March at the Forschungszentrum Jlich. Leading scientists from research and industry gave 220 students and young scientists from 25 countries and five continents a comprehensive overview of the interdisciplinary research field "Soft Matter" at the interface between physics, chemistry, biology and the life sciences. Soft matter is ubiquitous in a vast range of technological applications and is of fundamental relevance in such diverse fields as chemical, environmental, and food industry as well as life sciences. Over the past years, soft matter science has been largely extended in its scope from more traditional areas such as colloids and polymers to the study of biological systems, soft nanoscale materials, and the development of novel composites and microfluidic devices.

Soft and biological materials share fundamental structural and dynamical features including a rich variety of morphologies and non-equilibrium phenomena, self-organisation, an unusual frictiondominated flow dynamics, and a high sensitivity to external fields. These properties emerge from the cooperative interplay of many degrees of freedom, with spatio-temporal correlations that can span a huge range from nano- to millimetres and nanoseconds to days. The key requirements for the advancement in the field of these highly complex soft materials are: The development of novel experimental techniques to study properties of individual components in processes and the cooperative behavior of many interacting constituents. The synthesis of complex materials, self-organized and biomimetic systems with novel or unusual properties will broaden the spectrum of applications. The exploration of advanced theoretical and computer simulation methods that span the large range of time and length scales and allow to cope with an increasing complexity of molecular constituents. Existing methods need to be extended and new approaches are required to describe systems far from equilibrium, e.g., in life sciences and material processing. Structural and novel functional properties of soft and biological materials need to be studied invoking self-organization and hierarchical structure formation, entropic particle interactions and fluid-like aspects of biological materials such as vesicles and cells. The unusual dynamics of complex fluids requires special approaches to gain insight into diffusion transport properties, rheology and mesoscopic flow behavior, which are influenced by a delicate interplay of hydrodynamic interactions, thermal fluctuations, and external fields.

The IFF Spring School 2008 at the Forschungszentrum Jlich, Germany, addressed advanced experimental techniques and

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applications, and theoretical and computer simulation methods on an undergraduate and graduate student level. Introductory lectures provided the basis of important experimental and theoretical tools. More advanced lectures explained practical aspects of various methods and lead the participants from basic methods to the frontiers of current research. The lectures covered the following topics: Scattering Techniques Single Molecule Techniques Equilibrium- and Non-equilibrium Statistical Physics Microfluidics Computer Simulations Synthesis

Self-Organisation Flow Properties and Rheology Biomechanics Macromolecules and Colloids Membranes and Interfaces Biomimetic Systems Glasses and Gels

The school offered about 50 hours of lectures plus discussions, as well as the opportunity to participate in practical courses and visits to the participating institutes at the Forschungszentrum Jlich. The local media coverage included newspapers, radio and television.

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Jlich Soft Matter Days 2008

J.K.G. Dhont 1 , G. Gompper 2 , D. Richter 3
1 2 3

IFF-7: Soft Condensed Matter IFF-2: Theoretical Soft-Matter and Biophysics IFF-5: Neutron Scattering

The Jlich Soft Matter Days is a yearly conference, held at the Gustav-Stresemann-Institut in Bonn, Germany. The number of participants is limited to about 220, which ensures an informal atmosphere and avoids parallel sessions. The conference attracts scientists from all continents and has a pronounced international character. An important part of the conference are two posters sessions with more than 120 contributions.

The aim of these meetings is to bring together scientists from all soft matter disciplines and from biophysics. The systems of interest include colloidal dispersions, polymer-solutions, -mixtures and -melts, block copolymers, binary and ternary amphiphilic systems (microemulsions), membranes, vesicles and biological macromolecules. While many of these systems have already been investigated for a long period of time, only recently their common features and mixtures have come into focus. In addition, in recent years, systems with various types of complex particles have been studied, where the particles exhibit properties that are both of colloidal, polymeric and possibly amphiphilic character. For example, Janus colloids share features of colloids and amphiphiles, and fd-viruses are in between rod-like colloids and stiff polymers. These highly complex systems are characterized by structural units with typical length scales ranging from nanometers to micrometers. The experimental and theoretical investigations as well as the understanding of the properties of these materials poses enormous challenges due to their high complexity, the large number of cooperative degrees of freedom, and the large range of relevant length, time and energy scales. We hope that this conference provided a forum to share and discuss the latest advances for all researchers in this eld. The topics that have been discussed 2008 in the colloid session range from Archimedian tilings on quasicrystalline surfaces (C. Bechinger, Stuttgart), multiple glassy states in star-polymer mixtures (Ch.N. Likos, Dsseldorf), synthesis of nanoparticle-microgel composites (L.M. Liz-Marzan, Vigo), to buckling of microcapsules (A. Imhof, Utrecht), colloids at interfaces (W.K. Kegel, Utrecht) and dynamics of charged colloids (G. Ngele, Jlich).

FIG. 1: Announcement of the Jlich Soft Matter Days 2008.

In the polymer session, highlights include talks about capillary wrinkling of oating polymer lms (T.P. Russell, Amherst), amplication of tension in branched polymers (M. Rubinstein, Chapel Hill), the physical and biological signicance of transition in DNA (K. Yoshikawa, Kyoto), and the rheology of branched entangled polymer melts (D.J. Read, Leeds). In the session on physics of the cell, the main topics were the role of DNA conformations in gene control (R. Metzler, Mnchen), cell shapes and forces on patterned substrates (U. Schwarz, Karlsruhe), ber organization in living cells (F. Ndlec, Heidelberg),

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An important part of conference are the poster sessions. The posters were grouped according to the same topics as the oral contributions. The more than 120 poster contributions were split into two poster sessions, which gave all participants the opportunity for lively discussions at the posters, an impression of which is given in Fig.2. For more information about the 2008 conference and the upcoming 2009 meeting, please visit the conference webpage http://www.fz-juelich.de/iff/jsmd2008. We hope that the Jlich Soft Matter Days will continue to be successful as an inter-disciplinary meeting on soft matter and biophysics.

FIG. 2: Discussions during one of the two the poster session.

and bacterial swarming (J. Vermant, Leuven). The main interests in the session on proteins were on neutron scattering to obtain information on structure (D.I. Svergun, Hamburg) and inter-domain dynamics (R. Biehl, Jlich), NMR experiments and simulations on self-similar dynamics (G. Kneller, Orlans), and protein aggregation in chinese century eggs (E. Eiser, Cambridge). Some of the topics of interest in the session on self-assembly were the kinetics of block-copolymer micelles (R. Lund, San Sebastian) and lipid vesicles (M. Nakano, Kyoto), rheology of microphaseseparated diblock copolymers (T. Ohta, Kyoto), microscopic swimmers at surfaces (J. Elgeti, Jlich), and cytoskeleton dynamics of liposomes (C. Sykes, Paris). Finally, the session on hydrodynamics included talks on ordering of colloids by ow and sedimentation (M.J. Solomon, Ann Arbor), micro-uidic crystals (R. Bar-Ziv, Rehovot), and heat and mass transport at interfaces (J.-L. Barrat, Lyon).

FIG. 3: The participants meet in the central court of the Gustav-Stresemann-Institut in Bonn.

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Publications

2007 2008
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Institute of Solid State Research Theoretical Soft Matter and Biophysics (IFF-2) 2007
Belitsky, V.; Maric, N.; Schtz, G. M. Phase transition in a cellular automaton model of a highway on-ramp Journal of Physics A - Mathematical and General, 40 (2007), 11221 - 11243 Brzank, A.; Schtz, G. M. Phase transition in the two-component symmetric exclusion process with open boundaries Journal of Statistical Mechanics: Theory and Experiment, 8 (2007), P08028 Cherstvy, A. G. Effect of a Low-Dielectric Interior on DNA Electrostatic Response to Twisting and Bending Journal of Physical Chemistry B, 111 (2007), 12933 - 12937 Eisenriegler, E.; Bringer, A. Polymer depletion profiles around nonspherical colloidal particles Journal of Chemical Physics, 127 (2007), 034904 Gtze, I.; Noguchi, H.; Gompper, G. Relevance of angular momentum conservation in mesoscale hydrodynamics simulations Physical Review E, 76 (2007), 046705 Grosskinsky, S.; Schtz, G. M.; Willmann, R. D. Rigorous results on spontaneous symmetry breaking in a one-dimensional driven particle system Journal of Statistical Physics, 128 (2007), 587 - 606 Gwan, J. F.; Baumgaertner, A. Cooperative Transport in a Potassium Ion Channel Journal of Chemical Physics, 127 (2007), 045103 Gwan, J. F.; Baumgaertner, A. Ion Transport in a Nanochannel Journal of Computational and Theoretical Nanoscience : for all Theoretical and Computational Aspects in Science, Engineering, and Biology, 4 (2007), 50 - 56 Harris, R. J.; Schtz, G. M. Fluctuation theorems for stochastic dynamics Journal of Statistical Mechanics : Theory and Experiment, (2007), P07020 Harris, R. J.; Stinchcombe, R. B.* Scaling approach to related disordered stochastic and free-fermion models Physical Review E, 75 (2007), 031104 Kohyama, T.*; Gompper, G. Defect Scars on Flexible Surfaces with Crystalline Order Physical Review Letters, 98 (2007), 198101-1 - 198101-4 Noguchi, H.; Gompper, G. Swinging and Tumbling of Fluid Vesicles in Shear Flow Physical Review Letters, 98 (2007), 128103 Noguchi, H.; Gompper, G. Transport coefficients of dissipative particle dynamics with finite time step Europhysics Letters, 79 (2007), 36002 Noguchi, H.; Kikuchi, N.*; Gompper, G. Particle-based mesoscale hydrodynamic techniques Europhysics Letters, 78 (2007), 10005 140

Ripoll, M.; Winkler, R. G.; Gompper, G. Hydrodynamic screening of star polymers in shear flow European Physical Journal E, 23 (2007), 349 - 354 Schtz, G. M.; Harris, R.J.* Hydrodynamics of the Zero-range Process in the Condensation Regime Journal of Statistical Physics, 127 (2007) 2, 419 - 430 Winkler, R. G. Diffusion and segmental dynamics of rodlike molecules by fluorescence correlation spectroscopy Journal of Chemical Physics, 127 (2007), 054904 Winkler, R. G.; Cherstvy, A. G. Adsorption of Weakly Charged Polyelectrolytes onto Oppositely Charged Spherical Colloids Journal of Physical Chemistry B, 111 (2007), 8486 - 8493 Yang, Y.; Burkhardt, T. W.; Gompper, G. Free energy and extension of a semiflexible polymer in cylindrical confining geometries Physical Review E, 76 (2007), 011804

2008
Baumgaertner, A. Concepts in Bionanomachines: Translocators Journal of Computational and Theoretical Nanoscience : for all Theoretical and Computational Aspects in Science, Engineering, and Biology, 5 (2008) 9, 1852 - 1890 Cannavacciuolo, L.; Winkler, R. G.; Gompper, G. Mesoscale simulations of polymer dynamics in microchannel flows Europhysics Letters, 83 (2008), 34007-1 - 34007-6 Chatterjee, S.; Barma, M. Shock probes in a one dimensional Katz-Lebowitz-Spohn Model Physical Review E, 77 (2008), 061124-1 - 061124-8 Cherstvy, A. Protein - DNA interactions: Reaching and Recognizing the Targets Journal of Physical Chemistry B, 112 (2008), 4741 - 4750 Cherstvy, A. G. DNA Cholesteric Phases: The Role of DNA Molecular Chirality and DNA-DNA Electrostatic Interactions Journal of Physical Chemistry B, 112 (2008), 12585 - 12595 Finken, R.*; Lamura, A.*; Seifert, U.*; Gompper, G. Two-dimensional fluctuating vesicles in linear shear flow European Physical Journal E, 25 (2008), 309 - 321 Frank, S.; Winkler, R. G. Polyelectrolyte electrophoresis: Field effects and hydrodynamic interactions Europhysics Letters, 83 (2008), 38004 Gompper, G.; Dhont, J. K. G.; Richter, D. Editorial : A unified view of soft matter systems? European Physical Journal E, 26 (2008), 1 - 2 Grokinsky, S.; Schtz, G. M. Discontinuous Condensation Transition and Nonequivalence of Ensembles in a Zero-Range Process Journal of Statistical Physics, 132 (2008), 77 - 108 Grosskinsky, S.; Chleboun, P.; Schtz, G. M. Instability of condensation in the zero-range process with random interaction Physical Review E, 78 (2008) 3, 030101

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Harish, R.; Karevski, D.*; Schtz, G. M. Molecular traffic control for a cracking reaction Journal of Catalysis, 253 (2008), 191 199 Huang, C.C. Kinetics and dynamics of wormlike micelles under shear Europhysics Letters, 81 (2008), 58002 McWhirter, J.L. Phase behavior of a simple dipolar fluid under shear flow in an electric field Journal of Chemical Physics, 128 (2008), 034502 Noguchi, H.; Gompper, G. Transport coefficients of off-lattice mesoscale-hydrodynamics simulation techniques Physical Review E, 78 (2008), 016706-1 - 016706-12 Popkov, V.; Salerno, M.; Schtz, G. M. Asymmetric simple exclusion process with periodic boundary driving Physical Review E, 78 (2008) 1, 011122 Priezzhev, V.B.; Schtz, G. M. Exact solution of the Bernoulli matching model of sequence alignment Journal of Statistical Mechanics : Theory and Experiment, (2008), P09007 Ripoll, M.; Holmqvist, P.; Winkler, R. G.; Gompper, G.; Dhont, J. K. G.; Lettinga, M. P. Attractive Colloidal Rods in Shear Flow Physical Review Letters, 101 (2008), 168302-1 - 168302-4 Ripoll, M.; Winkler, R. G.; Mussawisade, K.; Gompper, G. Mesoscale hydrodynamics simulations of attractive rod-like colloids in shear flow Journal of Physics: Condensed Matter, 20 (2008), 404209 Schtz, G. M.; Brandaut, M.; Trimper, S. Exact solution of a stochastic susceptible-infectious-recovered model Physical Review E, 78 (2008), 061132 Tao, Y.-G.; Gtze, I.O.; Gompper, G. Multiparticle collision dynamics modeling of viscoelastic fluids Journal of Chemical Physics, 128 (2008), 144902-1 - 144902-12 Verberck, B.; Heresanu, V.; Rouziere, S.; Cambedouzou, J.; Launois, P.; Kovats, E.; Pekker, S.; Vliegenthart, G.; Michel, K.H.; Gompper, G. Fullerene-cubane: X-ray Scattering Experiments and Monte Carlo Simulations Fullerenes Nanotubes and Carbon Nanostructures, 16 (2008), 293 - 300 Vliegenthart, G.; Gompper, G. Mechanical properties of icosahedral virus capsids Journal of Computer-Aided Materials Design, 14 (2007), 111 - 119 Yang, Y.; Elgeti, J.; Gompper, G. Cooperation of Sperm in Two Dimensions: Synchronization, Attraction and Aggregation through Hydrodynamic Interactions Physical Review E, 78 (2008), 061903

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Institute of Solid State Research Neutron Scattering (IFF) 2007

Allgaier, J.; Willbold, S.; Chang, T. Synthesis of Hydrophobic Poly(alkylene oxide)s and Amphiphilic Poly(alkylene oxide) Block Copolymers Macromolecules, 40 (2007) 3, 518 - 525 Bta, A.*; Varga, Z.*; Goerigk, G. Biological systems as nanoreactors: Anomalouls small-angle scattering study of the CdS nanoparticle formation in multilamellar vesicles Journal of Physical Chemistry B, 111 (2007) 8, 1911 - 1915 Bta, A.*; Varga, Z.*; Goerigk, G. Vesicles as reactors of nanoparticles: an anomalous small-angle X-ray scattering study of the domains rich in copper ions Journal of Applied Crystallography, 40 (2007), s259 - s263 Buchenau, U.; Wischnewski, A.; Ohl, M.; Fabiani, E.* Neutron scattering evidence on the nature of the boson peak Journal of Physics: Condensed Matter, 19 (2007), 205106 Buchenau, U.; Zorn, R.; Ohl, M.; Wischnewski, A. Dielectric and thermal relaxation in the energy landscape Philosophical Magazine, 87 (2007) 3/5, 389 - 400 Chang, J.*; Pailhs, S.*; Shi, M.*; Mnsson, M.*; Claesson, T.*; Tjernberg, O.*; Voigt, J.; Perez, V.*; Patthey, L.*; Momono, N.*; Oda, M.*; Ido, M.*; Schnyder, A.*; Mudry, C.*; Mesot, J.* When low- and high-energy electronic responses meet in in cuprate superconductors Physical Review B, 75 (2007), 224508-1 - 224508-6 De Luca, E.*; Waigh, T. A.*; Monkenbusch, M.; Kim, J. S.*; Jeon, H. S.* Neutron spin echo study of the dynamics of micellar solutions of randomly sulphonated polystyrene Polymer, 48 (2007), 3930 - 3934 Faulhaber, E.*; Stockert, O.*; Schmalzl, K.; Jeevan, H.S.*; Deppe, M.*; Geibel, C.*; Steglich, F.*; Loewenhaupt, M.* Spatial separation of antiferromagnetism and superconductivity in CeCu2Si2 Journal of Magnetism and Magnetic Materials, 310 (2007), 295 - 297 Feygenson, M.; Kentzinger, E.; Ziegenhagen, N.; Rcker, U.; Goerigk, G.; Wang, Y.*; Brckel, T. Contrast variation by anomalous X-ray scattering applied to investigation of the interface morphology in a giant magnetoresistance Fe/Cr/Fe trilayer Journal of Applied Crystallography, 40 (2007) 3, 532 - 538 Frank, C.; Frielinghaus, H.; Allgaier, J.; Prast, H. Nonionic Surfactants with Linear and Branched Hydrocarbon Tails: Compositional Analysis, Phase Behavior, and Film Properties in Bicontinuous Microemulsions Langmuir, 23 (2007) 12, 6526 - 6535 Frick, B.*; Koza, M.*; Zorn, R. Editorial European Physical Journal Special Topics: ST, 141 (2007) Frielinghaus, H. Small-angle scattering model for multilamellar vesicles Physical Review E, 76 (2007), 051603-1 051603-8 Goerigk, G.; Huber, K.*; Schweins, R.* Probing the extent of the Sr2+ ion condensation to anionic polyacrylate coils: A quantitative anomalous small-angle x-ray scattering study Journal of Chemical Physics, 127 (2007), 154908-1 - 154908-8 143

Goll, G.*; Stockert, O.*; Prager, M.; Yoshino, T.*; Takabatake, T.* Low energy excitations in CeBiPt Journal of Magnetism and Magnetic Materials, 310 (2007), 1773 Gutirrez, J.*; Barandiarn, J. M.*; Bermejo, F. J.*; Mondelli, C.*; Romano, P.*; Fouquet, P.*; Monkenbusch, M. Evidence for two disparate spin dynamic regimes within Fe-substituted La0,7 Pb0,3)Mn1-xFex)O3 (0 kleiner/gleich x kleiner/gleich 0,2) colossal magentoresistive manganites: Neutron spin-echo measurements Physical Review B, 76 (2007) 18, 184401 Holderer, O.; Frielinghaus, H.; Monkenbusch, M.; Allgaier, J.; Richter, D.; Farago, B.* Hydrodynamic effects in bicontinuous microemulsions measured by inelastic neutron scattering European Physical Journal E, 22 (2007), 157 - 161 Iatrou, H.*; Frielinghaus, H.; Hanski, S.*; Ferderigos, N.*; Ruokolainen, J.*; Ikkala, O.*; Richter, D.; Mays, J.*; Hadjichristidis, N.* Architecturally induced multiresponsive vesicles from well-defined polypeptides. Formation of gene vehicles Biomacromolecules, 8 (2007) 7, 2173 - 2181 Ibberson, R. M.*; Prager, M. Temperature-dependent crystal structure analysis of methyl iodide by high-resolution neutron powder spectroscopy Zeitschrift fr Kristallographie, 222 (2007), 416 Ioffe, A.; Bodnarchuk, V.; Bussmann, K.; Mueller, R. Larmor labeling by time-gradient magnetic fields Physica B: Condensed Matter, 397 (2007), 108 - 111 Kentzinger, E.; Frielinghaus, H.; Rcker, U.; Ioffe, A.; Richter, D.; Brckel, T. Probing lateral magnetic nanostructures by polarized GISANS Physica B: Condensed Matter, 397 (2007), 43 - 46 Kirstein, O.*; Prager, M.; Grimm, H.; Buchsteiner, A.*; Wischnewski, A. Quasielastic neutron scattering experiments including activation energies and mathematical modeling of methyl halide dynamics Journal of Chemical Physics, 127 (2007), 094504 Klose,F.*; Holtkamp,N.*; Richter,D. Die Megawatt-Spallationsneutronenquelle SNS: Neue Chancen fr die Erforschung der kondensierten Materie Physik Journal, 1 (2007),23 Laurati, M.; Stellbrink, J.; Lund, R.; Willner, L.; Richter, D.; Zaccarelli, E.* A system with star-like morphology and interactions Physical Review E, 76 (2007), 041503 Lebedev, D.V.*; Monkenbusch, M.; Shalguev, V.I.*; Lantsov, V.A.*; Isaev-Ivanov, V. V. Dynamic properties of RecA protein filaments from E-coli and P-aeruginosa investigated by neutron spin echo Biofizika, 52 (2007) 5, 799 - 803 Li, H. F.; Su, Y.; Peron, J.; Meuffels, P.; Walter, J. M.; Skowronek, R.; Brckel, T. Neutron-diffraction study of structural transition and magnetic order in orthorhombic and rhombohedral La7/8Sr1/8Mn1gammaO3+delta Journal of Physics: Condensed Matter, 19 (2007), 176226-1 -176226-12 Li, H.; Su, Y.; Peron, J.; Meuffels, P.; Walter, J.*; Skowronek, R.*; Brckel, T. Correlation between structural and magnetic properties of La7/8Sr1/8Mn1-gammaO3 delta with controlled nonstoichiometry Journal of Physics: Condensed Matter, 19 (2007), 016003-1 - 016003-12 Lund, R.; Willner, L.; Richter, D.; Iatrou, H.*; Hadjichristidis, N.*; Lindener, P.* Unraveling the equilibrium chain exchange kinetics of polymeric micelles using small-angle neutron scattering archtectural and topological effects Journal of Applied Crystallography, 40 (2007), s327 - s331 Maccarrone, S.; Frielinghaus, H.; Allgaier, J.; Richter, D.; Lindner, P.* SANS Study of Polymer-Linked Droplets Langmuir, 23 (2007) 19, 9559 - 9562 Mangiapia, G.; Ricciardi, R.*; Auriemma, F.*; de Rosa, C.*; Lo Celso, F.*; Triolo, R.*; Heenan, R. K.*; Radulescu, A.; Tedeschi, A. M.*; D'Errico, G.*; Paduano, L.* Mesoscopic and Microscopic Investigation on Poly(vinyl alcohol) Hydrogels in the Presence of Sodium Decylsulfate Journal of Physical Chemistry B, 111 (2007) 9, 2166 - 2173 144

Mattern, N.*; Gemming, T.*; Goerigk, G.; Eckert, J.* Phase separation in amorphous Ni-Nb-Y alloys Scripta Materialia, 57 (2007), 29 - 32 Monkenbusch, M.; Richter, D. High resolution neutron spectroscopy - a tool for the investigation of dynamics of polymers and soft matter Comptes Rendus Physique, 8 (2007), 845 - 864 Niedzwiedz, K.; Wischnewski, A.; Monkenbusch, M.; Richter, D.; Genix, A.-C.*; Arbe, A.*; Colmenero, J.*; Strauch, M.*; Straube, E.* Polymer Chain Dynamics in a Random Environment: Heterogeneous Mobilities Physical Review Letters, 98 (2007), 168301 Oberdisse, J.*; Hine, P.*; Pyckhout-Hintzen, W. Structure of interacting aggregates of silicia nanoparticles in a polymer matrix: Small-angle scattering and reverse Monte-Carlo simulations Soft Matter, 3 (2007) 2, 476 - 485 Prager, M.; Desmedt, A.*; Allgaier, J.; Russina, M.*; Jansen, E.*; Natkaniec, I.*; Pawlukojc, A.*; Press, W. Methyl group rotation and whole molecule dynamics in methyl bromide hydrate Phase Transitions, 80 (2007) 6/7, 473 Prager, M.; Pawlukojc, A.*; Wischnewski, A.; Wuttke, J. Inelastic neutron scattering study of methyl groups rotation in some methylxanthines Journal of Chemical Physics, 127 (2007) 214509 Prager, M.; Sawka-Dobrowolska, W.*; Sobczyk, L.*; Pawlukojc, A.*; Grech, E.*; Wischnewski, A.; Zamponi, M. X-ray diffraction and inelastic neutron scattering study of 2,6-dimethylpyrazine chloranilic acid complex Chemical Physics, 332 (2007), 1 - 9 Prager, M.; Wischnewski, A.; Bator, G.*; Grech, E.*; Pawlukojc, A.*; Sobczyk, L.* INS spectroscopic study of the 1:1 tetramethylpyrazine (TMP) squaric acid (H(2)SQ) complex Chemical Physics, 334 (2007), 148 - 153 Pranzas, P. K.*; Dornheim, M.*; Bsenberg, U.*; Fernandez, J. R. A.*; Goerigk, G .; Roth, S. V.*; Gehrke, R.*; Schreyer, A.* Small-angle scattering investigations of magnesium hydride used as a hydrogen storage material Journal of Applied Crystallography, 40 (2007) Suppl. 1, s383 - s387 Radulescu, A.; Schwahn, D.; Stellbrink, J. Hierarchical structures formed by partially crystalline polymers in solution: from fundamentals to applications - a combined conventional, focusing and ultra-small-angle neutron scattering study Journal of Applied Crystallography, 40 (2007), s97 - s100 Richter, D. Polymer dynamics: from synthetic polymers to proteins Journal of Applied Crystallography, 40 (2007), s28 - s33 Rother, G.*; Melnichenko, Y. B.*; Cole, D. R.*; Frielinghaus, H.; Wignall, G. D.* Microstructural characterization of adsorption and depletion regimes of supercritical fluids in nanopores Journal of Physical Chemistry B, 111 (2007) 43, 15736 - 15742 Rubio-Retama, J.*; Tamimi, F. M.*; Heinrich, M.; Lopez-Cabarcos, E.* Synthesis and Characterization of Poly(magnesium acrylate) Microgels Langmuir, 23 (2007) 16, 8538 - 8543 Rzhevsky, A. A.*; Krichevtsov, B. B.*; Su, Y.; Schneider, C. M. Probing the (001) surface of magnetite crystals by second harmonic generation Journal of Physics: Condensed Matter, 19 (2007), 396006-1 - 396006-10 Schmalzl, K. Volume and pressure dependence of ground-state and lattice dynamical properties of BaF2 from density-functional methods Physical Review B, 75 (2007), 014306-1 - 014306-11 Schmalzl, K.; Strauch, D.* Static Pockels constants pij and pijk of CaF2 and BaF2 under strain from ab initio calculations Physical Review B, 76 (2007), 205106-1 - 205106-8 145

Schnhals, A.; Schick, C.; Frick, B.*; Mayorova, M.; Zorn, R. Molecular Dynamics in Glass-forming Poly(phenyl methyl siloxane) as investigated by Broadband Thermal, Dielectric and Neutron Spectroscopy Journal of Non-Crystalline Solids, 353 (2007), 3853 - 3861 Schnhals, A.*; Schick, Ch.*; Huith, H.*; Frick, B.*; Mayorova, M.; Zorn, R. Molecular Mobility of Poly (phenyl methyl siloxane) Investigated by Thermal, Dielectric and Neutron Spectroscopy Polymeric Materials Science and Engineering, 97 (2007), 948 - 949 Schwahn, D.; Ma, Y.*; Clfen, H.* Mesocrystal to Single Crystal Transformation of D,L-Alanine Evidenced by Small Angle Neutron Scattering Journal of Physical Chemistry C, 111 (2007) 8, 3224 - 3227 Schweika, W.; Hermann, R.; Prager, M.; Peron, J.; Keppens, V.* Dumbbell rattling in thermoelectric zinc antimony Physical Review Letters, 99 (2007), 125501-1 - 125501-4 Shi, Q.*; Voss, J.*; Jacobsen, H.S.*; Lefmann, K.*; Zamponi, M.; Vegge, T.* Point defect dynamics in sodium aluminium hydrides- A combined quasielastic neutron scattering and density functional theory study Journal of Alloys and Compounds, 469 (2007), 446 - 447 Sinibaldi, R.*; Ortore, M. G.*; Spinozzi, F.*; Carsughi, F.*; Frielinghaus, H.; Cinelli, S.*; Onori, G.*; Mariani, P.* Preferential hydration of lysozyme in water/glycerol mixtures: A small-angle neutron scattering study Journal of Chemical Physics, 126 (2007) 23, 235101-1 - 235101-9 Spinozzi, F.*; Mariani, P.*; Saturni, L.*; Carsughi, F.; Bernstorff, S.*; Cinelli, S.*; Onori, G.* Met-myoglobin Association in Dilute Solution during Pressure-Induced Denaturation: an Analysis at pH 4.5 by HighPressure Small-Angle X-ray Scattering Journal of Physical Chemistry B, 111 (2007), 3822 - 3830 10.1021/jp063427m Steffens, P.*; Sidis, Y.*; Link, P.*; Schmalzl, K.; Nakatsuji, S.*; Maeno, Y.*; Braden, M.* Field-Induced Paramagnons at the Metamagnetic Transition of Ca1.8Sr0.2RuO4 Physical Review Letters, 99 (2007), 217402-1 217402-4 Stellbrink, J.; Niu, A.; Allgaier, J.; Richter, D.; Koenig, B. W.*; Hartmann, R.*; Coates, G. W.*; Fetters, L. J.* Analysis of Polymeric Methylaluminoxane (MAO) via Small Angle Neutron Scattering Macromolecules, 40 (2007), 4972 - 4981 Szekely, N. K.*; Almasy, L.*; Radulescu, A.; Rosta, L.* Small-angle neutron scattering study of aqueous solutions of pentanediol and hexanediol Journal of Applied Crystallography, 40 (2007) Suppl. 1, s307 - s311 Tehei, M.*; Franzetti, B.*; Wood, K.*; Gabel, F.*; Fabiani, E.*; Jasnin, M.*; Zamponi, M.; Oesterhelt, D.*; Zaccai, G.*; Ginzburg, M.*; Ginzburg, B. Z.* Neutron scattering reveals extremely slow cell water in a Dead Sea organism Proceedings of the National Academy of Sciences of the United States of America, 104 (2007), 766 - 771 Vaccaro, M.*; Accardo, A.*; D'Errico, G.*; Schilln, K.*; Radulescu, A.; Tesauro, D.*; Morelli, G.*; Paduano, L.* Peptides and Gd Complexes Containing Colloidal Assemblies as Tumor-Specific Contrast Agents in MRI: Physicochemical Characterization Biophysical Journal, 93 (2007), 1736 - 1746 Vainio, U.*; Pirkkalainen, K.*; Kisko, K.*; Goerigk, G.; Kotelnikova, N. E.*; Serimaa, R.* Copper and copper oxide nanoparticles in a cellulose support studied using anomalous small-angle x-ray scattering European Physical Journal D, 42 (2007), 93 - 101 Varga, Z.*; Bta, A.*; Goerigk, G. Localization of dihalogenated phenols in vesicle systems determined by contrast variation X-ray scattering Journal of Applied Crystallography, 40 (2007) , s205 - s208 Voigt, J.; Persson, J.; Kim, J. W.*; Bihlmayer, G.; Brckel, Th. Strong coupling between the spin polarization of Mn and Tb in multiferroic TbMnO3 determined by x-ray resonance exchange scattering Physical Review B, 76 (2007), 104431-1 - 104431-5

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Voss, J.*; Jacobson, H.S.*; Zamponi, M.; Lefmann, K.*; Vegge, T.*; Shi, Q.* Hydrogen dynamics in Na3AIH6: A combined density functional theory and quasielastic neutron scattering study Journal of Physical Chemistry B, 111 (2007), 3886 Walter, W.*; Borlein, M.*; Eyelein, F.*; Gehring, M.*; Kozielewski, T.; Kramer, A.*; Monkenbusch, M.; Ohl, M.; Paul, A.; Schrauth, B.*; Tiemann, Ch. Design of a pair of superconducting solenoids for a neutron spin-echo spectrometer at the SNS IEEE Transactions on Applied Superconductivity, 17 (2007) 2, 1209 - 1212 Zhang, F.*; Skoda, M. W. A.*; Jacobs, R. M. J.*; Zorn, S.*; Martin, R. A.*; Martin, C. M.*; Clark, G. F.*; Goerigk, G.; Schreiber, F.* Gold Nanoparticles Decorated with Oligo(ethylene glycol) Thiols: Protein Resistance and Colloidal Stability Journal of Physical Chemistry B, 111 (2007) 49, 12229 - 12237 Zorn, R. Multiple scattering correction of neutron scattering elastic scans Nuclear Instruments and Methods in Physics Research Section A, 572 (2007), 874 881

2008
Arbe, A.*; Genix, A.-C.*; Colmenero, J.*; Richter, D. Anomalous Relaxation od Self Assembled Alkyl-Nanodomains in higher order Poly(n-Alkyl Metacrylates) Soft Matter, 4 (2008) 9, 1792 - 1795 Auriemma, F.*; De Rosa, C.*; Ricciardi, R.*; Lo Celso, F.*; Triolo, R.*; Pipich, V. Time-Resolving Analysis of Cryotropic Gelation of Water/Poly(vinyl alcohol) Solutions via Small-Angle Neutron Scattering Journal of Physical Chemistry B, 112 (2008) 3, 816 - 823 Biehl, R.; Hoffmann, B.; Monkenbusch, M.; Falus, P.*; Prost, S.*; Merkel, R.; Richter, D. Direct Observation of Correlated Interdomain Motion in Alcohol Dehydrogenase Physical Review Letters, 101 (2008), 138102-1 - 138102-4 Birczynski, A.*; Ylinen, E.E.*; Punkkinen, M.*; Prager, M.; Szymocha, A.M.*; Lalowicz, Z.T.* Deuteron NMR relaxation, spectra, and evidence for the order-disorder phase transition in (ND4)2PtCl6 Journal of Chemical Physics, 128 (2008), 184510 Bodnarchuk,V. I.*; Kraan,W. H.*; Rekveldt,M. T.*; Ioffe,A. Neutron spin turners with a rotating magnetic field: first experiments Measurement Science and Technology, 19 (2008), 034012 Bosak, A.*; Schmalzl, K.; Krisch, M.*; van Beek, W.*; Kolobanov, V.* Lattice dynamics of beryllium oxide: Inelastic x-ray scattering and ab initio calculations Physical Review B, 77 (2008), 224303-1 - 224303-7 Bose, P.*; Mittal, R.; Choudhury, N.*; Chaplot, S. L.* Inelastic neutron scattering and lattice dynamics of ZrO2, Y2O3 and ThSiO4 Pramana : Journal of Physics, 71 (2008) 5, 1141 - 1146 Bta, A.*; Varga, Z.*; Goerigk, G. Structural description of the nickel part of a raney-type catalyst by using anomalous small-angle x-ray scattering Journal of Physical Chemistry C, 112 (2008) 12, 4427 - 4429 Brckel, T.; Kentzinger, E.; Mattauch, S.; Paul, A.; Rcker, U.; Voigt, J. A deeper look into magnetic nanostructures using advanced scattering methods Pramana : Journal of Physics, 71 (2008) 5, 895 Buchenau, U.; Schober, H.R. An atomic mechanism for the boson peak in metallic glasses Philosophical Magazine, 88 (2008) 33/35, 3885 3900 147

Burrows, H.D.*; Knaapila, M.*; Monkman, A.P.*; Tapia, M.J.*; Fonseca, S.M.*; Ramos, M.L.*; Pyckhout-Hintzen, W.; Pradhan, S.*; Scherf, U.* Structural Sudies on Cationic Poly9,9- bis [N,N,N-trimethylammonium) Alkyl] Fluorence-Co-1,4-Phenyleneiodides in Aqueous Solutions in the Presence of the Noninioic Surfactant Pentaethyleneglycol Monododecyl Ether (C12E5) Journal of Physics: Condensed Matter, 20 (2008), 104210 Chatterji, T.; Henry, P. F.*; Mittal, R.; Chaplot, S. L.* Negative thermal expansion of ReO3: Neutron diffraction experiments and dynamical lattice calculations Physical Review B, 78 (2008), 134105-1 - 134105-6 Chatterji, T.; Schneider, G. J.; Galera, R. M.* Low-Energy Nuclear Spin Excitations in NdMg3 and NdCo2 Physical Review B, 78 (2008), 012411-1 - 012411-4 Chen, J.*; Jung, P.; Hoffelner, W.*; Ullmaier, H. Dislocation loops and bubbles in oxide dispersion strengthened ferritic steel after helium implantation under stress Acta Materialia, 56 (2008), 250 - 258 Chen, J.*; Jung, P.; Pouchon, M. A.*; Rebac, T.*; Hoffelner, W.* Irradiation creep and precipitation in a ferritic ODS steel under helium implantation Journal of Nuclear Materials, 373 (2008), 22 - 27 Conrad, H.; Brckel, T.; Schfer, W.*; Voigt, J. POWTEX - the high-intensity time-of-flight diffractometer at FRM II for structure analysis of polycrystalline materials Journal of Applied Crystallography, 41 (2008), 836 - 845 Delcea, M.*; Krastev, R.*; Gutberlet, T.; Pum, D.*; Sleytr, U. B.*; Toca-Herrera, J.L.* Thermal stability, mechanical properties and water content of bacterial protein layers recrystallized on polyelectrolyte multilayers Soft Matter, 4 (2008), 1414 - 1421 Disch, S.; Cheetham, A. K.*; Ruschewitz, U.* Formation of unsaturated C3 hydrocarbons by the protolysis of magnesium sesquicarbide with ammonium halides Inorganic Chemistry, 47 (2008), 969 - 973 Ehlers, G.*; Mamontov, E.*; Zamponi, M.; Faraone, A.*; Qiu, Y.*; Cornelius, A. L.*; Booth, C.H.*; Kam, K.C.*; LeToquin, R.*; Cheetham, A. K.*; Gardner, J. S.* Frustrated spin correlations in diluted spin ice Ho_2-x La_x Ti_2 O_7 Journal of Physics: Condensed Matter, 20 (2008), 235206 Foster, L.J.*; Schwahn, D.; Pipich, V.; Holden, P.J.*; Richter, D. SANS characterization of polyhydroxyalkanoates and their PEgylated hybrids in solution Biomacromolecules, 9 (2008), 314 - 320 Frank, C.*; Frielinghaus, H.; Allgaier, J.; Richter, D. Hydrophilic Alcohol Ethoxylates as Efficiency booster for Microemulsions Langmuir, 24 (2008), 6036 Genix, A.C.*; Arbe, A.*; Arres-Igor, S.*; Colmenero, J.*; Richter, D.; Frick, B.*; Deen, P.* Neutron scattering and dielectric investigation of a dilute blend of poly(ethyleneoxide) in polyethersulfone Journal of Chemical Physics, 128 (2008), 184901 Goel, P.*; Mittal, R.; Chaplot, S. L.*; Tyagi, A. K.* Lattice dynamics of strontium tungstate Pramana : Journal of Physics, 71 (2008) 5, 1135 1139 Goldman, A. I.*; Argyriou, D. N.*; Ouladdiaf, B.*; Chatterji, T.; Kreyssig, A.*; Nandi, S.*; Ni, N.*; Bud'ko, S. L.*; Canfield, P. C.*; McQueeney, R. J.* Lattice and magnetic instabilities in CaFe2As2: A single crystal neutron diffraction study Physical Review B, 78 (2008), 100506(R) Gossuin, Y.*; Hocq, A.*; Vuong, Q. L.*; Disch, S.; Hermann, R. P.; Gillis, P.* Physico-chemical and NMR relaxometric characterization of gadolinium hydroxide and dysprosium oxide nanoparticles Nanotechnology, 19 (2008), 475102-1 - 475102-8 Gray, D. L.*; Long, G. J.*; Grandjean, F.*; Hermann, R. P.; Ibers, J. A.* Syntheses, Structure, and a Mssbauer and Magnetic Study of Ba4Fe2I5S4 Inorganic Chemistry, 47 (2008) 1, 94 100 148

Gupta, M.*; Gupta, A.*; Stahn, J.*; Gutberlet, T. Ordering and self-diffusion in FePt alloy film New Journal of Physics, 10 (2008), 053031 Holderer, O.; Monkenbusch, M; Borchert, G.*; Breunig, C.*; Zeitelhack, K.* Layout and performance of the polarizing guide system for the J-NSE spectrometer at the FRM II Nuclear Instruments and Methods in Physics Research Section A, 586 (2008) 1, 90 - 94 Holderer, O.; Monkenbusch, M.; Schtzler, R.; Kleines, H.; Westerhausen, W.; Richter, D. The JCNS neutron spin-echo spectrometer J-NSE at the FRM II Measurement Science and Technology, 19 (2008), 034022 Hu, R.*; Thomas, K. J.*; Lee, Y.*; Vogt, T.*; Choi, E. S.*; Mitrovic, V. F.*; Hermann, R. P.; Grandjean, F.*; Canfield, P. C.*; Kim, J. W.*; Goldman, A. I.*; Petrovic, C.* Colossal positive magnetoresistance in a doped nearly magnetic semiconductor Physical Review B, 77 (2008), 085212-1 - 085212-5 Hller, A.*; Prager, M.; Press, W.*; Seydel, T. Phase III of solid methane: The orientational potential and rotational tunneling Journal of Chemical Physics, 128 (2008), 034503 Ioffe, A. A new neutron spin-echo technique with time-gradient magnetic fields Nuclear Instruments and Methods in Physics Research Section A, 586 (2008) 1, 31 - 35 Ioffe, A.; Bodnarchuk, V.*; Bussmann, R.; Mller, R.*; Georgii, R.* A new neutron spin-echo spectrometer with time-gradient magnetic fields: First experimental test Nuclear Instruments and Methods in Physics Research Section A, 586 (2008) 1, 36 - 40 Kapnistos, M.*; Lang, M.*; Vlassopoulos, D.*; Pyckhout-Hintzen, W.; Richter, D.; Cho, D.*; Chang, T.*; Rubinstein, M.* Unexpected power-law stress relaxation of entangled ring polymers Nature Materials, 7 (2008), 997 - 1002 Karatas, Y.*; Pyckhout-Hintzen, W.; Zorn, R.; Richter, D.; Wiemhfer, H.-D.* SANS Investigation and Conductivity of Pure and Salt-Containing Poly(bismethoxy-phosphazene) Macromolecules, 41 (2008), 2212 - 2218 Kentzinger, E.; Rcker, U.; Toperverg, B.; Ott, F.*; Brckel, T. Depth-resolved investigation of the lateral magnetic correlations in a gradient nanocrystalline multilayer Physical Review B, 77 (2008), 104435-1 - 104435-19 Kimber, S. A. J.*; Argyriou, D. N.*; Yokaichiya, F.*; Habicht, K.*; Gerischer, S.*; Hansen, T.*; Chatterji, T.; Klingeler, R.*; Hess, C.*; Behr, G.*; Kondrat, A.*; Bchner, B.* Magnetic ordering and negative thermal expansion in PrFeAsO Physical Review B, 78 (2008), 140503-1 - 140503-4(R) Kolasinska, M.*; Krastev, R.*; Gutberlet, T.; Warszynski, P.* Swelling and Water Uptake of PAH/PSS Polyelectrolyte Multilayers Progress in Colloid and Polymer Science, 134 (2008), 30 - 38 Lebedev, D.V.*; Filatov, M.V.*; Kuklin, A. I.*; Islamov, A. K.*; Stellbrink, J.; Pantina, R.A.*; Denisov, Y.Y.*; Toperverg, B. P.*; Isaev-Ivanov, V. V.* Structural hierarchy of chromatin in chicken erythrocyte nuclei based on small-angle neutron scattering: Fractal nature of the large-scale chromatin organization Crystallography Reports, 53 (2008), 110 - 115 Li, Y.-L.*; Maurel, M.-C.*; Ebel, C.*; Vergne, J.*; Pipich, V.; Zaccai, G.* Self-association of adenine-dependent hairpin ribozymes European Biophysics Journal : with Biophysics Letters, 37 (2008) 2, 173 - 182 Lott, D.*; Fenske, J.*; Schreyer, A.*; Mani, P.*; Mankey, G. J.*; Klose, F.*; Schmidt, W.; Schmalzl, K.; Tartakovskaya, E. V.* Antiferromagnetism in a Fe50Pt40Rh10 thin film investigated using neutron diffraction Physical Review B, 78 (2008), 174413-1 - 174413-10 Lonetti, B.; Kohlbrecher, J.*; Willner, L.; Dhont, J. K. G.; Lettinga, M. P. Dynamic response of block copolymer wormlike micelles to shear flow Journal of Physics: Condensed Matter, 20 (2008), 404207 149

Manoshin, S.*; Ioffe, A. New modules for the VITESS software package: Time-gradient magnetic fields and neutron refractive lenses Nuclear Instruments and Methods in Physics Research Section A, 586 (2008) 1, 81 - 85 Mamontov, E.*; Zamponi, M.; Hammons, S.*; Keener, W.S.*; Hagen, M.*; Herwig, K.W.* BASIS: A new backscattering spectrometer at the SNS Neutron News, 19 (2008), 22 - 24 Mazumdar, C.*; Rotter, M.*; Frontzed, M.*; Michor, H.*; Doerr, M.*; Kreiyssig, A.*; Koza, M.*; Hiess, A.*; Voigt, J.; Behr, G.*; Gupta, L.C.*; Prager, M.; Loewenhaupt, M.* Crystalline Electric Field Effects in PrNi2B2C: Inelastic Neutron Scattering Physical Review B, 78 (2008), 144422 McGuire, M. A.*; Christianson, A. D.*; Sefat, A. S.*; Sales, B. C.*; Lumsden, M. D.*; Jin, R.*; Payzant, E. A.*; Mandrus, D.*; Luan, Y.*; Keppens, V.*; Varadarajan, V.*; Brill, J. W.*; Hermann, R. P.; Sougrati, M. T.*; Grandjean, F.*; Long, G.* Phase transitions in LaFeAsO: Structural, magnetic, elastic, and transport properties, heat capacity and Mssbauer spectra Physical Review B, 78 (2008), 094517-1 - 094517-10 Mishra, S. K.*; Mittal, R.; Choudhury, N.*; Chaplot, S. L.*; Pandey, D.* Inelastic neutron scattering and lattice dynamics of NaNbO3 and Sr0.70Ca0.30TiO3 Pramana : Journal of Physics, 71 (2008) 5, 1141 - 1146 Proceedings of the International Symposium on Neutron Scattering (15. - 18.01.2008); Pramana - Journal of Physics 71, (2008) 5, 1129 - 1134; ISSN 0304-4289; 2008 Indian; Academy of Sciences, Bangalore Mittal, R. Negative thermal expansion in framework compounds Pramana : Journal of Physics, 71 (2008) 4, 829 - 835 Mittal, R.; Su, Y.; Rols, S.*; Tegel, M.*; Chaplot, S. L.*; Schober, H.*; Chatterji, T.; Johrendt, D.*; Brueckel, Th. Phonon dynamics in Sr[sub 0.6]K[sub 0.4]Fe[sub 2]As[sub 2] and Ca[sub 0.6]Na[sub 0.4]Fe[sub 2]As[sub 2] from neutron scattering and lattice-dynamical calculations Physical Review B, 78 (2008) 22, 224518-1 - 224518-5 Mittal, R.; Su, Y.; Chatterji, T.*; Chaplot, S.L.*; Schober, H.; Rotter, M.*; Johrendt, D.*; Brckel, T. Inelastic neutron scattering and lattice-dynamical calculations of BaFe2As2 Physical Review B, 78 (2008), 104514 Narros, A.*; Arbe, A.*; Alvarez, F.*; Colmenero, J.*; Richter, D. Atomic motions in the alpha-beta merging regime of 1-4 polybutadiene: A molecular dynamics simulation study Journal of Chemical Physics, 128 (2008), 224905 Niedzwiedz, K.; Wischnewski, A.; Pyckhout-Hintzen, W.; Allgaier, J.; Richter, D.; Faraone, A.* Chain Dynamics and Viscoelastic Properties of Poly(ethylene-oxide) Macromolecules, 41 (2008), 4866 Oh, I.-H.*; Mattauch, S.; Heger, G.*; Lee, C.E.* Neutron Diffraction Study of a Rb0.5Tl0.5H2PO4 Single Crystal Journal of the Physical Society of Japan, 77 (2008) 9, 094602-1 - 094602-4 Papagiannopoulos, A.*; Fernyhough, C.M.*; Waigh, T. A.*; Radulescu, A. Scattering Study of the Structure of Polystyrene Sulfonate Comb Polyelectrolytes in Solution Macromolecular Chemistry and Physics, 209 (2008), 2475 - 2486 Pipich, V.; Willner, L.; Schwahn, D. The A-B Diblock copolymer as non-ordering external field in a three component A/B/A-B polymer blend (Part of the "Karl Freed Festschrift") Journal of Physical Chemistry B, 112 (2008) 50, 16170 - 16181 Pipich, V.; Balz, M.*; Wolf, S.E.*; Tremel, W.*; Schwahn, D. Nucleation and Growth of CaCO3 Mediated by the Egg-White Protein Ovalbumin: A Time-Resolved in-situ Study Using Small-Angle Neutron Scattering Journal of the American Chemical Society, 130 (2008) 21, 6879 - 6892 Prager, M.; Desmedt, A.*; Unruh, T.*; Allgaier, J. Dynamics and adsorption sites of guest molecules in methyl chloride hydrate Journal of Physics: Condensed Matter, 20 (2008), 125219

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Prager, M.; Grimm, H.; Natkaniec, I.*; Nowak, D.*; Unruh, T.* The dimensionality of ammonium reorientation in (NH4)2S2O8: the view from neutron spectroscopy Journal of Physics: Condensed Matter, 20 (2008), 125218 Radulescu, A.; Fetter, L.J.*; Richter, D. Polymer driven wax crystal control using partially crystalline polymeric materials Advances in Polymer Science, 210 (2008), 1 - 100 Radulescu, A.; Ioffe, A. Neutron guide system for small-angle neutron scattering instruments of the Jlich Centre for Neutron Science at the FRM-II Nuclear Instruments and Methods in Physics Research Section A, 586 (2008) 1, 55 - 58 Ramzi, A.*; Rijcken, C.*; Veldhuis, T.*; Schwahn, D.; Hennink, W.*; van Nostrun, C.* Core-Shell Structure of Degradable, Thermosensitive Polymeric Micelles studfied by Small-Angle Neutron Scattering Journal of Physical Chemistry B, 112 (2008) 3, 784 - 792 Schmalzl, K.; Strauch, D.*; Hiess, A.*; Berger, H.* Temperature dependent phonon dispersion in 2H-NbSe2 investigated using inelastic neutron scattering Journal of Physics: Condensed Matter, 20 (2008), 104240-1 - 104240-3 Schmidt, H.*; Gupta, M.*; Gutberlet, T.; Stahn, J.*; Bruns, M.* How to measure atomic diffusion processes in the sub-nanometer range Acta Materialia, 56 (2008), 464 - 470 Schneider, G. J.; Kerscher, M.; Gritz, D.* Messung der Volumennderung von Elastomeren bei hohen Dehnraten = Measurement of Volume Changes of Elestomeres at High Strain Rates Kautschuk Gummi Kunststoffe, 6 (2008) , 317 - 321 Schober, H.*; Farhi, E.*; Mezei, F.*; Allenspach, P.*; Andersen, K.*; Bentley, P.M.*; Christiansen, P.*; Cubitt, B.*; Heenan, R. K.*; Kulda, J.*; Langan, P.*; Lefmann, K.*; Lieutenant, K.*; Monkenbusch, M.; Willendrup, P.*; Saroun, J.*; Tindesmans, P.*; Zsigmond, G.* Tailored instrumentation for long-pulse neutron spallation sources Nuclear Instruments and Methods in Physics Research Section A, 589 (2008) 1, 34 46 Sinko, K.*; Huesing, N.*; Goerigk, G.; Peterlik, H.* Nanostructure of gel-derived aluminosilicate materials Langmuir, 24 (2008) 3, 949 - 956 Sougrati, M. T.*; Hermann, R. P.; Grandjean, F.*; Long, G. J.*; Brck, E.*; Tegus, O.*; Trung, N. T.*; Buschow, K. H. J.* A structural, magnetic, and Mssbauer spectral study of the magnetocaloric Mn1.1Fe0.9P1-xGex compounds Journal of Physics: Condensed Matter, 20 (2008), 475206-1 - 475206-9 Stellbrink, J.; Lonetti, B.; Rother, G.*; Willner, L.; Richter, D. Shear induced structures of soft colloids: Rheo-SANS experiments on kinetically frozen PEP-PEO diblock copolymer micelles Journal of Physics: Condensed Matter, 20 (2008), 404206 Stelzer, H.; Weissbacher, C.; Soltner, H.; Janssen, F.; Butzek, M.; Kozielewski, T.; Lindenau, B.; Monkenbusch, M.; Ohl, M. Investigation of the temperature rise due to eddy currents in large chopper disks operated at polarized neutron beamlines Nuclear Instruments and Methods in Physics Research Section A, 594 (2008) 2, 228 - 231 Strempfer, J.*; Hupfeld, D.; Voigt, J.; Bihlmayer, G.; Goldman, A. I.*; Brckel, Th. Resonant magnetic x-ray scattering from terbium Journal of Physics: Condensed Matter, 20 (2008), 445208-1 - 445208-7 Theis-Brhl, K.*; Westphalen, A.*; Zabel, H.*; Rcker, U.; McCord, J.*; Hink, V.*; Schmalhorst, J.*; Reiss, G.*; Weis, T.*; Engel, D.*; Ehresmann, A.*; Toperverg, B. P.* Hyper-domains in exchange bias micro-stripe pattern New Journal of Physics, 10 (2008), 093021-1 - 093021-21 Teixeira, S.C.M.*; Zaccai, G.*; Ankner, J.*; Bellissent-Funel, M.C.*; Bewley, R.*; Blakeley, M.P.*; Callow, P.*; Coates, L.*; Dahint, R.*; Dalgliesh, R.*; Dencher, N. A.*; Forsyth, V. T.*; Fragneto, G.*; Frick, G.*; Gilles, R.*; Gutberlet, T.; Haertlein, M.*; Hau, T.*; Huler, W.*; Heller, W.T.*; Herwig, K.*; Holderer, O.; Juranyi, F.*; Kampmann, R.*; Knott, R.*; Krueger, S.*; Langan, P.*; Lechner, R. E.*; Lynn, G.*; Majkrzak, C.*; May, R. P.*; Meilleur, F.*; Mo, Y.*; Mortensen, K.*; Myles, D.A.A.*; Natali, F.*; Neylon, C.*; Niimura, N.*; Ollivier, J.*; Ostermann, A.*; Peters, J.*; Pieper, J.*; Rhm, A.*; 151

Schwahn, D.; Shibata, K.*; Soper, A.K.*; Strssle, Th.*; Suzuki, J.*; Tanaka, I.*; Tehei, M.*; Timmins, P.*; Torikai, N.*; Unruh, T.*; Urban, V.*; Vavrin, R.*; Weiss, K.* New Sources and Instrumentation for Neutrons in Biology Chemical Physics, 345 (2008), 133 - 155 Varga, Z.*; Bta, A.*; Goerigk, G. Unbinding Transition in Lipid Multibilayers Induced by Copper(II) Ions Journal of Physical Chemistry B, 112 (2008) 29, 8430 8433 Xu, X. S.*; Angst, M.; Brinzari, T. V.*; Hermann, R. P.; Musfeldt, J. L.*; Christianson, A. D.*; Mandrus, D.*; Sales, B. C.*; McGill, S.*; Kim, J.-W.*; Islam, Z.* Charge order, dynamics, and magneto-structural transition in multiferroic LuFe2O4 Physical Review Letters, 101 (2008), 227602-1 227602-4 Yan, H.*; Frielinghaus, H.; Nykanen, A.*; Ruokolainen, J.*; Saiani, A.*; Miller, A.F.* Thermoreversible lysozyme hydrogels: properties and an insight into the gelation pathway Soft Matter, 4 (2008) 6, 1313 Zamponi, M.; Wischnewski, A.; Monkenbusch, M.; Willner, L.; Richter, D.; Falus, P.*; Farago, B.*; Guenza, M.G.* Cooperative dynamics in homopolymer melts: a comparision of theoretical predictions with neutron spin echo experiments Journal of Physical Chemistry B, 112 (2008), 16220 - 16229 Zelenak, V.*; Badanicova, M.*; Halamova, D.*; Cejka, J.*; Zukal, A.*; Murafa, N.*; Goerigk, G. Amine-modified ordered mesoporous silica: Effect of pore size on carbon dioxide capture Chemical Engineering Journal, 144 (2008) 2, 336 - 342 Zorn, R.; Mayorova, M.; Richter, D.; Frick, B.* Inelastic neutron scattering study of a glass-forming liquid in soft confinement Soft Matter, 4 (2008), 522 533

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Institute of Solid State Research Soft Matter (IFF-7) 2007

P. Ballesta, M. P. Lettinga, and S. Manneville Superposition rheology of shear-banding wormlike micelles J. Rheology (2007) Vol .51 (5), 1047-1072 Blochowicz, T*; Ggelein, C.; Spehr, T.*; Mller, M.*; Sthn, B.* Polymer-induced transient networks in water-in-oil microemusions studied by small-angle x-ray and dynamic light scattering Physical Review E, 76 (2007) 4, 041505 Dhont, J. K. G.; Wiegand, S.; Duhr, S.*; Braun, D.* Thermodiffusion of Charged Colloids: Single-Particle Diffusion Langmuir, 23 (2007), 1674 - 1683 Fan, T.-H.*; Dhont, J. K. G.; Tuinier, R. Motion of a sphere through a polymer solution Physical Review E, 75 (2007), 011803 Fan, T.-H.*; Xie, B.*; Tuinier, R. Asymptotic analysis of tracer diffusivity in nonadsorbing polmyer solutions Physical Review E, 76 (2007), 051405 Fleer, G. J.*; Skvortsov, A. M.*; Tuinier, R. A Simple Relation for the Concentration Dependence of Osmotic Pressure and Depletion Thickness in Polymer Solutions Macromolecular Theory and Simulations, 16 (2007), 531 - 540 Fleer, G. J.*; Tuinier, R. Analytical phase diagram for colloid-polymer mixtures Physical Review E, 76 (2007), 041802 Fleer, G. J.; Tuinier, R. The critical endpoint in phase diagrams of attractive hard spheres Physica A, 379 (2007), 52 - 58 Gapinski, J.; Patkowski, A.; Banchio, A. J.; Holmqvist, P.; Meier, G.; Lettinga, M. P.; Ngele, G. Collective diffusion in charge-stabilized suspensions: Concentration and salt effects Journal of Chemical Physics, 126 (2007), 104905 Holmqvist, P.; Dhont, J. K. G.; Lang, P. R. Colloidal dynamics near a wall studied by evanescent wave light scattering: Experimental and theoretical improvements and methodological limitations Journal of Chemical Physics, 126 (2007), 044707 Holmqvist, P.; Kleshchanok, D.; Lang, P. R. Unexpected slow near wall dynamics of spherical colloids in a suspension of rods Langmuir, 23 (2007), 12010 - 12015 Jungblut, S.*; Tuinier, R.; Binder, K.*; Schilling, T.* Depletion induced isotropic-isotropic phase separation in suspensions of rod-like colloids Journal of Chemical Physics, 127 (2007), 244909 K. Kang, S. Sprunt Light-Controlled Polymerization Kinetics for the Photostabilization of Cholesteric Fingerprint Rolls Mol.Cryst.Liq.Cryst., (2007) Vol. 466, pp. 23-38 Kang, K.; Wilk, A.*; Patkowski, A.*; Dhont, J. K. G. Diffusion of spheres in isotropic and nematic networks of rods: Electrostatic interactions and hydrodynamic screening Journal of Chemical Physics, 126 (2007), 214501 153

Kita, R.*; Polyakov, P.; Wiegand, S. Ludwig-Soret Effect of Poly(N-isopropylacrylamide): Temperature Dependence Study in Monohydric Alcohols Macromolecules, 40 (2007) 5, 1638 - 1642 Kleshchanok, D.; Lang, P. R. Steric Repulsion by Adsorbed Polymer Layers Studied with Total Internal Reflection Microscopy Langmuir, 23 (2007), 4332 -4339 Kohlbrecher, J.*; Bollhalder, A.*; Vavrin, R.*; Meier, G. A high pressure cell for small angle neutron scattering up to 500 MPa in combination with light scattering to investigate liquid samples Review of Scientific Instruments, 78 (2007), 125101 Lang, P. R. Depletion interaction mediated by polydisperse rods Journal of Chemical Physics, 127 (2007), 124906 Lettinga, M. P.; Grelet, E.* Self-diffusion of rodlike viruses through smectic layers Physical Review Letters, 99 (2007), 197802 McPhie, M. G.; Ngele, G. Long-time self-diffusion of charged colloidal particles: Electrokinetic and hydrodynamic interaction effects Journal of Chemical Physics, 127 (2007), 034906 Patkowski, A.*; Gapinski, J.*; Meier, G.; Kriegs, H. Isotropic Brillouin spectra of liquids having an internal degree of freedom Journal of Chemical Physics, 126 (2007), 014508 Polyakov, P.; Zhang, M.*; Mller-Plathe, F.*; Wiegand, S. Thermal diffusion measurements and simulations of binary mixtures of spherical molecules Journal of Chemical Physics, 127 (2007), 014502 Rediguieri, C.F.*; de Freitas, O.*; Lettinga, M. P.; Tuinier, R. Thermodynamic Incompatibility and Complex Formation in Pectin/Caseinate Mixtures Biomacromolecules, 8 (2007), 3345 - 3354 Rodriguez-Fernndez, J.; Perez-Juste, J.; Liz-Marzn, L.M.; Lang, P. R. Dynamic Light Scattering of Short Au Rods with Low Aspect Ratios Journal of Physical Chemistry C, 111 (2007), 5020 - 5025 Tuinier, R.; Taniguchi, T.*; Wensink, H. H.* Phase behavior of a suspension of hard sperocylinders plus ideal polymer chains European Physical Journal E, 23 (2007), 355 - 365 Wiegand, S.; Ning, H.; Kita, R.* Universal concentration dependence of the Soret coefficient in aqueous systems Journal of Non-Equilibrium Thermodynamics, 32 (2007) 3, 193 Wiegand, S.; Ning, H.; Kriegs, H. Thermal diffusion forced Rayleigh scattering setup optimized for aqueous mixtures Journal of Physical Chemistry B, 111 (2007), 14169 Zhang, Z.; Berns, A. E.; Willbold, S.; Buitenhuis, J. Synthesis of poly(ethylene glycol) (PEG)-grafted colloidal silica particles with improved stability in aqueous solvents Journal of Colloid and Interface Science, 310 (2007), 446 - 455 Zhang, Z.; Buitenhuis, J. Synthesis of Uniform Silica Rods, Curved Silica Wires, and Silica Bundles Using Filamentous fd Virus as a Template Small, 3 (2007), 3, 424 - 428

2008
Banchio, A. J.*; McPhie, M. G.; Ngele, G. Hydrodynamic and electrokinetic effects on the dynamics of charged colloids and macromolecules Journal of Physics: Condensed Matter, 20 (2008), 404213-1 - 404213-14 154

Banchio, A. J.*; Ngele, G. Short-time transport properties in dense suspensions: From neutral to charge-stabilized colloidal spheres Journal of Chemical Physics, 128 (2008), 104903 Blanco, P.*; Polyakov, P.; Bou-Ali, M.M.*; Wiegand, S. Thermal diffusion and molecular diffusion values for some alkane mixtures: a comparison between thermogravitational column and thermal diffusion forced Rayleigh scattering Journal of Physical Chemistry B, 112 (2008), 8340 - 8345 Dhont, J. K. G.; Briels, W. J.* Gradient and vorticity banding Rheologica Acta, 47 (2008), 257 - 281 Dhont, J. K. G.; Briels, W. J.* Single-particle thermal diffusion of charged colloids: Double-layer theory in a temperature gradient European Physical Journal E, 25 (2008), 61 - 76 Fleer, G. J.*; Tuinier, R. Analytical phase diagrams for colloids and non-adsorbing polymer Advances in Colloid and Interface Science, 143 (2008), 1 - 47 Ggelein, C.; Ngele, G.; Tuinier, R.; Gibaud, T.*; Stradner, A.*; Schurtenberger, P.* A simple patchy colloid model for the phase behavior of lysozyme dispersions Journal of Chemical Physics, 129 (2008), 085102 Grelet, E.*; Lettinga, M. P.; Bier, M.*; vanRoij, R.*; van der Schoot, P.* Dynamical and structural insights into the smectic phase of rod-like particles Journal of Physics: Condensed Matter, 20 (2008), 494213 Holmqvist, P.; Kleshchanok, D.; Lang, P. R. Interaction potential and near wall dynamics of spherical colloids in suspensions of rod-like fd-virus European Physical Journal E, 26 (2008), 177 - 182 Kang, K.; Dhont, J. K. G. Double-layer polarization induced transitions in suspensions of colloidal rods EPL : a Letters Journal Exploring the Frontiers of Physics, 84 (2008), 14005 Kang, K.; Lettinga, M. P.; Dhont, J. K. G. Is vorticity-banding due to an elastic instability? Rheologica Acta, 47 (2008), 499 - 508 Kleshchanok, D.; Tuinier, R.; Lang, P. R. Direct measurement of polymer-induced forces Journal of Physics: Condensed Matter, 20 (2008), 073101 Kriegs, H.; Meier, G.; Gapinski, J.*; Patkowski, A.* The effect of intramolecular relaxations on the damping of longitudinal and transverse phonons in polysiloxanes studied by Brillouin spectroscopy Journal of Chemical Physics, 128 (2008), 014507 Lonetti, B.; Kohlbrecher, J.*; Willner, L.; Dhont, J. K. G.; Lettinga, M. P. Dynamic response of block copolymer wormlike micelles to shear flow Journal of Physics: Condensed Matter, 20 (2008), 404207 McPhie, M.; Ngele, G. Nonmonotonic density dependence of the diffusion of DNA fragments in low-salt suspensions Physical Review E, 78 (2008), 060401(R) Meier, G.; Kriegs, H. A high pressure cell for dynamic light scattering up to 2kbars with conservation of plane of polarization Review of Scientific Instruments, 79 (2008), 013102 Meier, G.; Vavrin, R.*; Kohlbrecher, J.*; Buitenhuis, J.; Lettinga, M. P.; Ratajczyk, M.* SANS and dynamic light scattering to investigate the viscosity of toluene under high pressure up to 1800 bar Measurement Science and Technology, 19 (2008), 034017 Ning, H.; Datta, S.*; Sottmann, T.*; Wiegand, S. Soret effect of nonionic surfactants in water studied by different transient grating setups Journal of Physical Chemistry B, 112 (2008), 10927 - 10934 155

Ning, H.; Dhont, J. K. G.; Wiegand, S. Thermal-Diffusive Behavior of a Dilute Solution of Charged Colloids Langmuir, 24 (2008), 2426 - 2432 Nygard, K.*; Satapathy, D.K.*; Bunk, O.*; Diaz, A.*; Perret, E.*; Buitenhuis, J.; Pfeiffer, F.*; David, C.*; van der Veen, J.F.* Structure of confined fluids by y-ray interferometry using diffraction gratings Optics Express, 16 (2008) 25, 20522 - 20529 Patkowski, A.*; Gapinski, J.*; Fluerasu, A.*; Holmqvist, P.; Meier, G.; Lettinga, M. P.; Ngele, G. Structure and dynamics of colloidal suspensions studied by means of XPCS Acta Physica Polonica A, 114 (2008) 2, 339 - 350 Polyakov, P.; Wiegand, S. Systematic study of the thermal diffusion in associated mixtures Journal of Chemical Physics, 128 (2008), 034505 Polyakov, P.; Mller-Plathe, F.*; Wiegand, S. Reverse nonequilibrium molecular dynamics calculation of the Soret coefficient in liquid heptane/benzene mixtures Journal of Physical Chemistry B, 112 (2008) 47, 14999 - 15004 Ripoll, M.; Holmqvist, P.; Winkler, R. G.; Gompper, G.; Dhont, J. K. G.; Lettinga, M. P. Attractive Colloidal Rods in Shear Flow Physical Review Letters, 101 (2008), 168302-1 - 168302-4 Sprakel, J.*; Spruijt, E.*; Cohen Stuart, M.A.*; Besseling, N. A. M.*; Lettinga, M. P.; van der Gucht, J.* Shear banding and rheochaos in associative polymer networks Soft Matter, 4 (2008) 8, 1696 - 1705 Tuinier, R.; Fan, T.-H.* Scaling of nanoparticle retardation in semi-dilute polymer solutions Soft Matter, 4 (2008), 254 - 257 Tuinier, R.; Smith, A.*; Poon, W. C. K.*; Egelhaaf, S. U.*; Aarts, D.G.A.L.*; Lekkerkerker, H. N. W.*; Fleer, G. J.* Phase diagram for a mixture of colloids and polymers with equal size EPL : a Letters Journal Exploring the Frontiers of Physics, 82 (2008), 68002

156

Institute for Structural Biology and Biophysics Cellular Biophysics (ISB-1) 2007
Cukkemane, A.; Grter, B.; Novak, K.; Gensch, T.; Bnigk, W.; Gerharz, T.; Kaupp, U. B.; Seifert, R. Subunits act independently in a cyclic nucleotide-activated K+ channel EMBO Reports, 8 (2007) 8, 749 - 755 Dedecker, P.*; Muls, B.*; Hofkens, J.*; Enderlein, J.; Hotta, J.-I.* Orientational effects in the excitation and de-excitation of single molecules interacting with donut-mode laser beams Optics Express, 15 (2007) 6, 3372 - 3383 Dertinger, T.; Pacheco, V.*; von der Hocht, I.; Hartmann, R.*; Gregor, I.; Enderlein, J. Two focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements ChemPhysChem, 8 (2007) 3, 433 - 443 Eckel, R.*; Walhorn, V.*; Pelargus, C.*; Martini, J.*; Enderlein, J.; Nann, T.*; Anselmetti, D.*; Ros, R.* Fluorescence-emission control of single CdSe nanocrystals using gold-modified AFM tips Small, 3 (2007) 1, 44 - 49 Enderlein, J. Nucleotide specificity versus complex heterogeneity in exonuclease activity measurements Biophysical Journal, 92 (2007) 5, 5156 - 5158 Gensch, T.; Komolov, K. E.*; Senin, I. I.*; Philippov, P. P.*; Koch, K.-W.* Ca2+-dependent conformational changes in the neuronal Ca2+-sensor recoverin probed by the fluorescent dye Alexa647 Proteins - Structure Function and Bioinformatics, 66 (2007), 492 - 499 Gilbert, D.*; Franjic-Wrtz, C.*; Funk, K.*; Gensch, T.; Frings, S.*; Mhrlen, F.* Differential maturation of chloride homeostasis in primary afferent neurons of the somatosensory system International Journal of Developmental Neuroscience, 25 (2007), 479 - 489 Gregor, I.; Enderlein, J. Time-resolved methods in biophysics. 3. Fluorescence lifetime correlation spectroscopy Photochemical and Photobiological Sciences, 6 (2007), 13 - 18 Haber-Pohlmeier, S.; Abarca Heidemann, K.; Krschen, H. G.; Kaur Dhiman, H.*; Heberle, J.; Schwalbe, H.*; KleinSeetharaman, J.; Kaupp, U. B.; Pohlmeier, A. 2+ Binding of Ca to glutamic acid-rich polypeptides from the rod outer segment Biophysical Journal, 92 (2007), 3207 - 3214 Kapusta, P.*; Wahl, M.*; Benda, A.*; Hof, M.*; Enderlein, J. Fluorescence lifetime correlation spectroscopy Journal of Fluorescence, 17 (2007) 1, 43 - 48 Mataruga, A.; Kremmer, E.*; Mller, F. Type 3a and type 3b OFF cone bipolar cells provide for the alternative rod pathway in the mouse retina Journal of Comparative Neurology, 502 (2007), 1123 - 1137 Melnikov, S. M.*; Yeow, E. K. L.*; Uji-i, H.*; Cotlet, M.*; Mllen, K.*; De Schryver, F. C.*; Enderlein, J.; Hofkens, J.* Origin of simultaneous donor-acceptor emission in single molecules of peryleneimide-terrylenediimide labeled polyphenylene dendrimers Journal of Physical Chemistry B, 111 (2007) 4, 708 - 719 Mohrlder, J.; Stangler, T.; Hoffmann, Y.; Wiesehan, K.; Mataruga, A.; Willbold, D. Identification of calreticulin as a ligand of GABARAP by phage display screening of a peptide library FEBS Journal, 274 (2007) 5543 - 5555 Schrder-Lang, S.*; Schwrzel, M.*; Seifert, R.; Strnker, T.; Kateriya, S.*; Looser, J.*; Watanabe, M.*; Kaupp, U. B.; Hegemann, P.*; Nagel, G.* Fast manipulation of cellular cAMP level by light in vivo Nature Methods, 4 (2007), 39 - 42 157

Sykora, J.*; Kaiser, K.*; Gregor, I.; Bnigk, W.; Schmalzing, G.*; Enderlein, J. Exploring fluorescence antibunching in solution to determine the stoichiometry of molecular complexes Analytical Chemistry, 79 (2007) 11, 4040 - 4049 von der Hocht, I.; Enderlein, J. Fluorescence correlation spectroscopy in cells: Confinement and excluded volume effects Environmental and Molecular Pathology, 82 (2007) 2, 142-146 Wahl, M.*; Rahn, H.-J.*; Gregor, I.; Erdmann, R.*; Enderlein, J. Dead-time optimized time-correlated photon counting instrument with synchronized, independent timing channels Review of Scientific Instruments, 78 (2007), 033106 Zettl, H.*; Zettl, U.*; Krausch, G.*; Enderlein, J.; Ballauff, M.* Direct observation of single molecule mobility in semidilute polymer solutions Physical Review E, 75 (2007), 061804

2008
Funk, K.*; Woitecki, A.*; Franjic-Wrtz, C.*; Gensch, T.; Mhrlen, F.*; Frings, S.* Modulation of chloride homeostasis by inflammatory mediators in dorsal root ganglion neurons Molecular Pain, 4 (2008), 32 - 43 Harzheim, D.; Pfeiffer, K.H.; Fabritz, L.*; Kremmer, E.*; Buch, T.*; Waisman, A.*; Kirchhof, P.*; Kaupp, U. B.; Seifert, R. Cardiac pacemaker function of HCN4 channels in mice is confined to embryonic development and requires cyclic AMP EMBO Journal, 27 (2008), 692 - 703 Kaupp, U. B.; Kashikar, N.D.; Weyand, I. Mechanisms of sperm chemotaxis Annual Review of Physiology, 70 (2008), 93 - 117 Knop, G.C.; Seeliger, M.W.*; Thiel, F.; Mataruga, A.; Kaupp, U.B.; Friedburg, C.*; Tanimoto, N.*; Mller, F. Light response in the mouse retina are prolonged upon targeted deletion of the HCN1 channel gene European Journal of Neuroscience, 28 (2008) 2221 - 2230 Mach, T.*; Chimerel, C.*; Fritz, J.*; Fertig, N.*; Winterhalter, M.*; Ftterer, C. Miniaturized planar lipid bilayer: increased stability, low electric noise and fast fluid perfusion Analytical and Bioanalytical Chemistry, 390 (2008), 841 - 846 Whitehead, J.*; Vignjevic, D.*; Ftterer, C.; Beaurepaire, E.*; Robine, S.*; Farge, E.* Mechanical factors activate -catenin-dependent oncogene expression in APC1638N/+ mouse colon HFSP Journal, 2 (2008) 286 - 294

158

Institute for Structural Biology and Biophysics Molecular Biophysics (ISB-2) 2007
Berndt, A.*; Kottke, T.; Breitkreuz, H.*; Dvorsky, R.*; Hennig, S.*; Alexander, M.*; Wolf, E.* A Novel Photoreaction Mechanism for the Circadian Blue Light Photoreceptor Drosophila Cryptochrome Journal of Biological Chemistry, 282 (2007), 13011 13021 Gmelin, W.*; Zeth, K.*; Efremov, R.; Heberle, J.*; Tittor, J.*; Oesterhelt, D.* The Crystal Structure of the L1 Intermediate of Halorhodopsin at 1.9 Resolution Photochemistry and Photobiology, 83 (2007), 369 - 377 Gorshkova, Yu.E.; Gordeliy, V. I. Investigation of Interaction of Dimethylsulfoxide with Lipid Membranes via Small-Angle Neutron Scattering Crystallography Reports, 52 (2007), 535 - 539 Haber-Pohlmeier, S.; Abarca Heidemann, K.; Krschen, H. G.; Kaur Dhiman, H.*; Heberle, J.; Schwalbe, H.*; Klein-Seetharaman, J.; Kaupp, U. B.; Pohlmeier, A. Binding of Ca2+ to glutamic acid-rich polypeptides from the rod outer segment Biophysical Journal, 92 (2007), 3207 - 3214 Immeln, D.*; Schlesinger, R.; Heberle, J.*; Kottke, T.* Blue Light Induces Radical Formation and Autophosphorylation in the Light-sensitive Domain of Chlamydomonas Cryptochrome Journal of Biological Chemistry, 282 (2007) 30, 21720 - 21728 Majerus,T.*;Kottke,T.*;Laan,W.*;Hellingwerf,K.*; Heberle, J.* Time-resolved FT-IR spectroscopy traces signal relay within the blue-light receptor AppA. Chemphyschem., 12 (2007) 8, 1787-1789 Mennes, N.*; Klare, J. P.*; Chizhov, I.*; Seidel, R.*; Schlesinger, R.; Engelhard, M.* Expression of the halobacterial transducer protein HtrII from Natronomonas pharaonis in Escherichia coli FEBS Letters, 581 (2007) 7, 1487 1494 Mironova, O. S.; Budyak, I. L.; Bldt, G.; Schlesinger, R.; Heberle, J. FT-IR difference spectroscopy elucidates crucial interactions of sensory rhodopsin I with the cognate transducer Htrl Biochemistry, 46 (2007) 33, 9399 9405 Murugova, T. N.*; Gordeliy, V. I.; Kuklin, A. I*.; Solodovnikova, I.M.*; Yaguzhinsky, L. S.* Study of Three-Dimensionally Ordered Structures of Intact Mitochondria by Small-Angle Neutron Scattering Crystallography Reports, 52 (2007) 3, 521 - 524 Poetsch, A.*; Berzborn, R. J.*; Heberle, J.*; Link, T. A.*; Dencher, N. A.*; Seelert, H.* Biophysics and Bioinformatics Reveal Structural Differences of the Two Peripheral Stalk Subunits in Chloroplast ATP Synthase Journal of Biochemistry, 141 (2007), 411 - 420 Rogachev, A.V.; Cherniy, A.; Ozerin, A. N.; Gordeliy, V. I.; Kuklin, A. I. Spherical sector model for describing the experimental small-angle neutron scattering data for dendrimers Crystallography Reports, 52 (2007), 500 504 Skegro, D.*; Pulvermuller, A.*; Krafft, B.*; Granzin, J.; Hofmann, K. P.*; Bldt, G.; Schlesinger, R. N-terminal and C-terminal Domains of Arrestin Both Contribute in Binding to Rhodopsin (dagger) Photochemistry and Photobiology, 83 (2007), 385 - 393 Strucksberg, K.H.; Rosenkranz, T.; Fitter, J. Reversible and irreversible unfolding of multi-domain proteins Biochimica et Biophysica Acta, 1774 (2007), 1501 - 1603

159

2008
Budyak, I.L.*;Mironova, O.S.*;Yanamala, N.*;Manoharan,V.*;Bldt,G.; Schlesinger,R.; Klein-Seetharaman,J. Flexibility of the Cytoplasmic Domain of the Phototaxis Transducer II from Natronomonas pharaonis Journal of Biophysics, in press Jiang, X.*; Zaitseva, E.*; Schmidt, M.*; Siebert, F.*; Engelhard, M.*; Schlesinger, R.; Ataka, K.*; Vogel, R.*; Heberle, J.* Resolving voltage-dependent structural changes of a membrane photoreceptor by surface-enhanced IR difference spectroscopy Proceedings of the National Academy of Sciences of the United States of America, 105 (2008) 34, 12113 12117 Stadler, A.M.; Digel, I.*; Artmann, G.M.*; Embs, J.P.*; Zaccai, G.*; Bldt, G. Hemoglobin Dynamics in Red Blood Cells: Correlation to Body Temperature Biophysical Journal, 95 (2008) , 1-13 Thielmann, Y.; Weiergrber, O. H.; Mohrlder, J.; Koenig, B. W.; Hartmann, R.; Stangler, T.; Wiesehan, K.; Willbold, D. Characterization of the binding surface of the human protein GABARAP From Computational Biophysics to Systems Biology (CBSB08) : proceedings / ed.: U. H. E. Hansmann, J. H. Meinke, S. Mohanty, W. Nadler, O. Zimmermann. - Jlich, John von Neumann Institut fr Computing, 2008. - (NIC Series ; 40). - 978-3-9810843-6-8. Weiergrber, O. H.; Stangler, T.; Thielmann, Y.; Mohrlder, J.; Wiesehan, K.; Willbold, D. Ligand binding mode of GABA(A) receptor-associated protein Journal of Molecular Biology, 381 (2008), 1320 - 1331

160

Institute for Structural Biology and Biophysics Structural Biochemistry (ISB-3) 2007
Birkmann, E.; Henke, F.; Weinmann, N.; Dumpitak, C.; Groshup, M.; Funke, A.; Willbold, D.; Riesner, D. Counting of single prion particles bound to a capture-antibody surface (surface-FIDA) Veterinary Microbiology, 123 (2007) 294-304 Dertinger, T.; Pacheco, V.; von der Hocht, I.; Hartmann, R.; Gregor, I.; Enderlein, J. Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements ChemPhysChem, 8 (2007) 433-443 Elfrink, K.; Nagel-Steger, L.; Riesner, D. Interaction of the Cellular Prion Protein with Raft-like Lipid Membranes. Biological Chemistry, 388 (2007) 79-90. Funke, S. A.; Birkmann, E.; Henke, F.; Grtz, P.; Lange-Asschenfeldt, C.; Riesner, D.; Willbold, D. Single particle detection of amyloid- aggregates associated with Alzheimers disease Biochemical and Biophysical Research Communications, 364 (2007) 902-907. Hnel, K.; Willbold, D. SARS-CoV accessory protein 7a directly interacts with human LFA-1 Biological Chemistry, 388 (2007), 1325-1332. Hoffmann, S.; Jonas, E.; Knig, S.; Preusser-Kunze, A.; Willbold, D. Nef protein of human immunodeficiency virus type 1 binds its own myristoylated N-terminus Biological Chemistry, 388 (2007) 181-183 Leliveld, S. R.; Korth, C. The use of conformation-specific ligands and assays to dissect the molecular mechanisms of neurodegenerative diseases Journal of Neuroscience Research, 85 (2007) 2285-2297 Mohrlder, J.; Hoffmann, Y.; Stangler, T.; Hnel, K.; Willbold, D. Identification of clathrin heavy chain as a direct binding partner for the GABA type A receptor associated protein GABARAP Biochemistry, 46 (2007) 14537-14543. Mohrlder, J.; Stangler, T.; Wiesehan, K.; Hoffmann, Y.; Mataruga, A.; Willbold, D. Identification of calreticulin as ligand of GABARAP by phage display screening of a peptide library FEBS Journal, 274 (2007) 5543-5555 Muhs, A.; Hickmann, D. T.; Pihlgren, M.; Chuard, N.; Giriens, V.; Meerschman, C.; van der Auwera, I.; van Leuven, F.; Sugawara, M.; Weingertner, M. C.; Bechinger, B.; Greferath, R.; Kolonko, N.; Nagel-Steger, L.; Riesner, D.; Brady, R. O.; Pfeifer, A.; Nicolau, C. Liposomal vaccines with conformation-specific amyloid peptide antigens define immune response and efficacy in APP transgenic mice Proceedings of the National Academy of Sciences of the United States of America, 104 (2007) 23, 9810-9815 Nagel-Steger L, Demeler B, Willbold D, Aggregate size and shape distributions in amyloid- peptide solutions. NIC Workshop 2007, From Computational Biophysics to System Biology 2007. Eds. Ulrich. E. Hansmann, Jan Meinke, Sandipan Mohanty, Olav Zimmermann. NIC Series Volume 36, 235-237 (2007) Scheidt, H. A.; Vogel, A.; Eckhoff, A.; Knig, B. W.; Huster, D. Solid state NMR characterization of the putative membrane anchor of TWD1 from Arabidopsis thaliana European Biophysics Journal, 36 (2007) 393-404 Schmidt, H.; Hoffmann, S.; Tran, T.; Stoldt, M.; Stangler, T.; Wiesehan, K.; Willbold, D. Solution structure of a Hck SH3 domain ligand complex reveals novel interaction modes Journal of Molecular Biology, 365 (2007) 1517-1532 161

Schnke, S.; Novak, K.; Stoldt, M.; Kaupp, U. B.; Willbold, D. Resonance assignment of the cyclic nucleotide binding domain from a cyclic nucleotide-gated K+ channel in complex with cAMP Biomolecular NMR Assignments, 1 (2007) 179-181 Spiess, H. W.; Jeschke, G.; Knig, B. W.; Willbold, D. Magnetische Resonanzspektroskopie Nachrichten aus der Chemie, 55 (2007) 288-293 Stangler, T.; Tran, T.; Hoffmann, S.; Schmidt, H.; Jonas, E.; Willbold, D. Competitive displacement of full-length HIV-1 Nef from Hck SH3 domain by a high affinity artificial peptide Biological Chemistry, 388 (2007) 611-615 Stangler T, Hartmann R, Willbold D, Knig BW, Modern high resolution NMR for the study of structure, dynamics and interactions of biological macromolecules. in Progress in Physical Chemistry Vol. 1 / Different Aspects of Intermolecular Interaction, Oldenbourg, Mnchen (2007) Stellbrink, J.; Niu, A. Z.; Allgaier, J.; Richter, D.; Knig, B. W.; Hartmann, R.; Coates, G. W.; Fetters, L. J. Analysis of polymeric methylaluminoxane (MAO) via small angle neutron scattering Macromolecules, 40 (2007) 4972-4981 Wiesehan, K.; Funke, S. A.; Fries, M.; Willbold, D. Purification of recombinantly expressed and cytotoxic human amyloid-beta peptide Journal of Chromatography B, 856 (2007) 229-233 Wittlich, M.; Knig, B. W.; Hoffmann, S.; Willbold, D. Structural characterisation of the transmembrane and cytoplasmic domains of human CD4. Biochimica et Biophysica Acta: Biomembranes, 1768 (2007) 2949-2960 Wittlich, M.; Knig, B. W.; Willbold, D. Expression, purification, and membrane reconstitution of a CD4 fragment comprising the transmembrane and cytoplasmic domains of the receptor Protein Expression and Purification, 55 (2007) 198-207

2008
Bailly, A.; Sovero, V.; Vincenzetti, V.; Santelia, D.; Bartnik, D.; Knig, B. W.; Mancuso, S.; Martinoia, E.; Geisler, M. Modulation of P-glycoproteins by auxin transport inhibitors is mediated by interaction with immunophilins. Journal of Biological Chemistry 283 (2008) 21817-21826 Birkmann E.; Riesner D. Prion infection seeded fibrillization or more? Prion, 2 (2008) 67-72 Birkmann, E.; Henke, F.; Funke, S. A.; Bannach, O.; Riesner, D.; Willbold, D. A highly sensitive diagnostic assay for aggregate-related diseases e.g. prion disease and Alzheimer's disease. Rejuvenation Research, 11 (2008) 359-363 Elfrink, K.; Ollesch J.; Sthr J.; Willbold D.; Riesner D.; Gerwert K. Structural changes of PrPc upon membrane anchoring. Proceedings of the National Academy of Sciences of the United States of America 105 (2008) 10815-10819 Funke, S. A.; Birkmann, E.; Henke, F.; Grtz, P.; Lange-Asschenfeldt, C.; Riesner, D.; Willbold, D. An ultra sensitive assay for early diagnosis of Alzheimer's disease. Rejuvenation Research, 11 (2008) 315-318 Gardiennet, C.; Loquet, A.; Etzkorn, M.; Heise, H.; Baldus, M.; Bckmann, A. Structural constraints for the Crh protein from solid-state NMR experiments. Journal Biomolecular NMR 40 (2008) 239-250 Hartmann R, Stangler T, Koenig BW, Willbold D, Exploring protein-ligand interactions by solution NMR. in Proteomics Sample Preparation, J. v. Hagen, Wiley-VCH (2008)

162

Heise, H. Solid-State NMR Spectroscopy of Amyloid Proteins. ChemBioChem 9 (2008) 179-189 Heise, H.; Celej, M. S.; Becker, S.; Riedel, D.; Pelah, A.; Kumar, A.; Jovin, T. M.; Baldus, M. Solid-state NMR reveals structural differences between fibrils of wild-type and disease-related A53T mutant alpha-synuclein Journal of Molecular Biology, 380 (2008) 444-450 Kaimann, T.; Metzger, S.; Kuhlmann, K.; Brandt, B.; Birkmann, E.; Hltje, H. D.; Riesner, D. Molecular model of an alpha-helical prion protein dimer and its monomeric subunits as derived from chemical cross-linking and molecular modeling calculations. Journal of Molecular Biology, 376 (2008), 582-596 Leliveld, S. R.; Bader, V.; Hendriks, P.; Prikulis, I.; Sajnani, G.; Requena, J.R.; Korth, C. Insolubility of disrupted-in-schizophrenia 1 disrupts oligomer-dependent interactions with nuclear distribution element 1 and is associated with sporadic mental disease. Journal of Neuroscience, 28 (2008) 15, 3839-3845 Leliveld, S. R.; Stitz, L.; Korth, C. Expansion of the octarepeat domain alters the misfolding pathway but not the folding pathway of the prion protein Biochemistry, 47 (2008) 23, 6267-6278 Mller, C. B.; Loman, A.; Pacheco, V.; Koberling, F.; Willbold, D.; Richtering, W.; Enderlein, J. Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy. Europhysics Letters 83 (2008) 46001 Nagel-Steger L, Demeler B, Hochdrfer K, Schrader T, Willbold D, Modulation of aggregate size and shape distributions of amyloid- peptide solutions by a designed -sheet breaker. NIC Workshop 2007, From Computational Biophysics to System Biology CBSB08. Eds. Ulrich. E. Hansmann, Jan H. Meinke, Sandipan Mohanty, Walter Nadler, Olav Zimmermann. NIC Series Volume 40, 333-336 (2008) Panza, G.; Sthr, J.; Birkmann, E.; Riesner, D.; Willbold, D.; Baba, O.; Terashima, T.; Dumpitak, C. Aggregation and amyloid fibril formation of the prion protein is accelerated in presence of glycogen. Rejuvenation Research 11 (2008) 365-369 Panza, G.; Sthr, J.; Dumpitak, C.; Papathanassiou, D.; Weinmann, N.; Wei, J.; Riesner, D.; Willbold, D.; Birkmann, E. Spontaneous and BSE-prion-seeded amyloid formation of full length recombinant bovine Prion Protein. Biochemical and Biophysical Research Communications 373 (2008) 493-497 Sthr, J.; Weinmann, N.; Wille, H.; Kaimann, T.; Nagel-Steger, L.; Birkmann, E.; Panza, G.; Prusiner, S. B.; Eigen, M.; Riesner, D Mechanisms of prion protein assembly into amyloid. Proceedings of the National Academy of Sciences of the United States of America, 105 (2008) 2409-2414 Thielmann, Y.; Mohrlder, J.; Knig, B. W.; Stangler, T.; Hartmann, R.; Hoeltje, H. D.; Willbold, D. An indole binding site major determinant of the ligand specificity of the GABA type A receptor associated protein GABARAP. ChemBioChem, 9 (2008) 1767-1775 Thielmann Y, Weiergrber OH, Mohrlder J, Koenig BW, Hartmann R, Stangler T, Wiesehan K, Willbold D. Characterization of the binding surface of the human protein GABARAP. NIC Workshop 2007, From Computational Biophysics to System Biology CBSB08. Eds. Ulrich. E. Hansmann, Jan H. Meinke, Sandipan Mohanty, Walter Nadler, Olav Zimmermann. NIC Series Volume 40, 401-403 (2008) van Groen, T.; Wiesehan, K.; Funke, S. A.; Kadish, I.; Nagel-Steger, L.; Willbold, D. Reduction of Alzheimer's Disease Amyloid Plaque Load in Transgenic Mice by D3, a D-enantiomeric Peptide Identified by Mirror Image Phage Display. ChemMedChem, 3 (2008) 1848-1852 Weiergrber, O. H.; Stangler, T.; Thielmann, Y.; Mohrlder, J.; Wiesehan, K.; Willbold, D. Ligand binding mode of GABA(A) receptor-associated protein Journal of Molecular Biology, 381 (2008), 1320-1331 Wiesehan, K.; Sthr, J.; Nagel-Steger, K.; van Groen, T.; Riesner, D.; Willbold, D. Inhibition of cytotoxicity and amyloid fibil formation by a D-amino acid peptide that specifically binds to Alzheimers disease amyloid peptide Protein Engineering Design and Selection, 21 (2008), 241-246 163

Wittlich, M.; Knig, B. W.; Willbold, D. Structural consequences of phosphorylation of two serine residues in the cytoplasmic domain of HIV-1 VpU. Journal of Peptide Science, 14 (2008) 804-810

164

Institute of Bio- and Nanosystems Bioelectronics (IBN-2) 2007

Akram, R.*; Eker, T.*; Bozbey, A.*; Fardmanesh, M.*; Schubert, J.; Banzet, M. Effect of rf Pumping Frequency and rf Input Power on the Flux to Voltage Transfer Function of rf-SQUIDs Applied Superconductivity IEEE Transactions on Applied Superconductivity, 17 (2007) 2, 676 - 679 Barannik, A. A.*; Cherpak, N. T.*; Prokopenko, Yu. V.*; Filipov, Yu. F.*; Shaforost, E.N.; Shipilova, I. A.* Two-layered disk quasi-optical dielectric resonators: electrodynamics and application perspectives for complex permittivity measurements of lossy liquids Measurement Science and Technology, 18 (2007) 7, 2231 - 2238 Belyaev, A. E.*; Naumov, A. V.*; Tarasov, G. G.*; Komarov, A. V.*; Tacano, M.*; Danylyuk, S. V.; Vitusevich, S. A. Nature of low-energy optical emission in doped AlGaN/GaN heterostructures Journal of Applied Physics, 101 (2007), 033709-1 - 033709-5 Cesa, C. M.; Kirchgener, N.; Mayer, D.; Schwarz, U. S.*; Hoffmann, B.; Merkel, R. Micropatterned silicon elastomer for high resolution analysis of cell force patterns Review of Scientific Instruments, 78 (2007) 3, 034301 Christiaens, P.*; Abouzar, M. H.; Poghossian, A.; Wagner, T.; Bijnens, N.*; Williams, O. A.*; Daenen, W. H.*; Haenen, K.*; Douheret, O.*; DHaen, J.*; Mekhalif, Z.*; Schning, M. J.; Wagner, P.* pH sensitivity of nanocrystalline diamond films Physica Status Solidi A, 204 (2007) 9, 2925 - 2930 Danilchenko, B. A.*; Zelensky, S. E.*; Drok, E. A.*; Belyaev, A. E.*; Kochelap, V. A.*; Lth, H.; Vitusevich, S. A. Enhancement by electric field of high-speed photoconductivity in AlGaN/GaN heterostructures Applied Physics Letters, 90 (2007) 15, 152102-1 - 152102-3 Fu, Z.*; Wu, A.*; Vilarinho, P. M.*; Kingon, A. I.*; Wrdenweber, R. Low dielectric loss BaNd2Ti5O14 thick films prepared by an electrophoretic deposition technique Applied Physics Letters, 90 (2007), 052912 Gasteier, P.*; Reska, A.; Schulte, P.; Salber, J.*; Offenhusser, A.; Moeller, M.*; Groll, J.* Surface Grafting of PEO-Based Star-Shaped Molecules for Bioanalytical and Biomedical Applications Macromolecular Bioscience, 7 (2007) 8, 1010 - 1023 Gonzlez, M. P.; Hollmann, E.; Wrdenweber, R. Quantitative analysis of the guidance of vortices in superconducting films with magnetic dots Journal of Applied Physics, 102 (2007), 063904 He, M.; Klushin, A. M.; Klein, N. Meandering Bicrystal Josephson Junction arrays in a hemispherical Fabry-Perot Resonator Superconductor Science and Technology, 20 (2007), s413 - s418 He, M.; Klushin, A. M.; Yan, S. L.*; Klein, N. Optimization of Bicrystal Josephson Junctions and Arrays in a Fabry-Perot Resonator IEEE Transactions on Applied Superconductivity, 17 (2007) 2, 934 - 937 Hollmann, E.; Wrdenweber, R. Analysis of Defects in Epitaxial Oxide Thin Films via X-Ray Diffraction Technology Thin Solid Films, 515 (2007) 7/8, 3530 Ingebrandt, S.; Han, Y.; Nakamura, F.*; Poghossian, A.; Schning, M. J.; Offenhusser, A. Label-free detection of single nucleotide polymorphisms utilizing the differential transfer function Biosensors and Bioelectronics, 22 (2007), 2834 - 2840 Jung, A.; Gronewold, T. M. A.*; Tewes, M.*; Quandt, E.*; Berlin, P. Biofunctional structural design of SAW sensor chip surfaces in a microfluidic sensor system Sensors and Actuators B, 124 (2007), 46 - 52 165

Jung, A.; Wolters, B.; Berlin, P. (Bio)functional surface structural design of substrate materials based on self-assembled monolayers from aminocellulose derivatives and amino(organo)polysiloxanes Thin Solid Films, 515 (2007) 17, 6867 - 6877 Krause, H.-J.; Wolters, N.; Zhang, Y.; Offenhusser, A.; Miethe, P.*; Meyer, M. H. F.*; Hartmann, M.*; Keusgen, M.* Magnetic particle detection by frequency mixing for immunoassay applications Journal of Magnetism and Magnetic Materials, 311 (2007), 436 - 444 Kuzel, P.*; Kadlec, F.*; Petzelt, J.*; Schubert, J.; Panaitov, G. Highly tunable SrTiO3 thin film structures for applications in the terahertz range Applied Physics Letters, 91 (2007), 232911 Meszaros, G.; Kronholz, S.; Karthuser, S.; Mayer, D.; Wandlowski, Th. Electrochemical Fabrication and Characterization of Nanocontacts and nm-sized Gaps Applied Physics A, 87 (2007), 569 - 575 Meyburg, S.*; Stockmann, R.; Moers, J.; Offenhusser, A.; Ingebrandt, S. Advanced CMOS process for floating gate field-effect transistors in bioelectronic applications Sensors and Actuators B, 128 (2007), 208 - 217 Meyer, M. H. F.*; Hartmann, M.*; Krause, H.-J.; Blankenstein, G.*; Mller-Chorus, B.*; Oster, J.*; Miethe, P.*; Keusgen, M. CRP determination based on a novel magnetic biosensor Biosensors and Bioelectronics, 22 (2007), 973 - 979 Meyer, M. H. F.*; Krause, H.-J.; Hartmann, M.*; Miethe, P.*; Oster, J.*; Keusgen, M.* Francisella tularensis detection using a magnetic labels and a magnetic biosensor based on frequency mixing Journal of Magnetism and Magnetic Materials, 311 (2007), 259 - 263 Meyer, M. H. F.*; Stehr, M.*; Bhuju, S.*; Krause, H.-J.; Hartmann, M.*; Miethe, P.*; Singh, M.*; Keusgen, M.* Magnetic biosensor for the detection of Yersinia pestis Journal of Microbiological Methods, 68 (2007), 218 - 224 Offenhusser, A.; Bcker-Meffert, S.; Decker, T.; Helpenstein, R.; Gasteier, P.*; Groll, J.*; Mller, M.*; Reska, A.; Schfer, S.; Schulte, P.; Vogt-Eisele, A.* Microcontact printing of proteins for neuronal cell guidance Soft Matter, 3 (2007) 3, 290 - 298 Pabst, M.; Wrobel, G.; Ingebrandt, S.; Sommerhage, F.; Offenhusser, A. Solution of the Poisson-Nernst-Planck equations in the cell-substrate interface European Physical Journal E, 24 (2007), 1 - 8 Petrychuk, M. V.*; Belyaev, A. E.*; Kurakin, A. M.; Danylyuk, S. V.; Klein, N.; Vitusevich, S. Mechanisms of current formation in resonant tunneling AlN/GaN heterostructures Applied Physics Letters, 91 (2007) 22, 222112 Poghossian, A.; Abouzar, M. H.; Amberger, F.; Mayer, D.; Han, Y.; Ingebrandt, S.; Offenhusser, A.; Schning, M. J. Field-effect sensors with charged macromolecules: characterization by capacitance-voltage, constant-capacitance, impedance-spectroscopy and atomic-force microscopy methods Biosensors and Bioelectronics, 22 (2007), 2100 - 2107 Poghossian, A.; Ingebrandt, S.; Abouzar, M. H.; Schning, M. J. Label-free detection of charged macromelecules by using a field-effect-based sensor platform: Experiments and possible mechanisms of signal generation Applied Physics A, 87 (2007), 517 - 524 Qiu, L.; Zhang, Y.; Krause, H.-J.; Braginski, A. I.; Burghoff, M.*; Trahms, L.* Nuclear magnetic resonance in the earth's magnetic field using a nitrogen-cooled superconducting quantum interference device Applied Physics Letters, 91 (2007), 072505 Qiu, L.; Zhang, Y.; Krause, H.-J.; Braginski, A. I.; Usoskin, A.* High-temperature superconducting quantum interference device with cooled LC resonant circuit for measuring alternating magnetic fields with improved signal-to-noise ratio Review of Scientific Instruments, 78 (2007), 054701 Schning, M. J.; Abouzar, M. H.; Poghossian, A.; Han, Y.; Offenhusser, A.; Ingebrandt, S. Markierungsfreie DNA-Detektion mit Silizium-Feldeffekt-Sensoren - Messeffekte oder Artefakte? Technisches Messen, 74 (2007) 9, 466 - 474 166

Schning, M. J.; Kloock, J. P. About 20 years of silicon-based thin-film sensors with chalcogenide glass materials for heavy metal analysis: Technological aspects of fabrication and miniaturization (Review) Electroanalysis, 19 (2007) 19/20, 2029 - 2038 Sosso, A.*; Andreone, D.*; Lacquaniti, V.*; Klushin, A. M.; He, M.; Klein, N. Metrological Study of YBCO Josephson Junction Arrays Integrated in a Fabry-Perot Resonator IEEE Transactions on Applied Superconductivity, 17 (2007) 2, 874 - 877 Turek, M.; Ketterer, L.*; Claen, M.*; Berndt, H. K.*; Elbers, G.*; Krger, P.*; Keusgen, M.*; Schning, M. J. Development and electrochemical investigations on an EIS- (electrolyte-insulator-semiconductor) based biosensor for cyanide detection Sensors, 7 (2007), 1415 - 1426 Wagner, T.; Maris, R. J.*; Ackermann, H.-J.*; Otto, R.; Beging, S.; Poghossian, A.; Schning, M. J. Handheld measurement device for field-effect sensor structures: Practical evaluation and limitations Sensors and Actuators B, 127 (2007) , 217-223 Wagner, T.; Molina, R.; Yoshinobu, T.*; Kloock, J. P.; Biselli, M.*; Canzoneri, M.*; Schnitzler, T.*; Schning, M. J. Handheld multi-channel LAPS device as a transducer platform for possible biological and chemical multi-sensor applications Electrochimica Acta, 53 (2007), 305 - 311 Wrdenweber, R.; Hollmann, E.; Kutzner, R.; Schubert, J. Induced Ferroelectricity in Strained Epitaxial SrTiO3 Films on Various Substrates Journal of Applied Physics, 102 (2007), 044119-1 - 044119-5 Wrdenweber, R.; Hollmann, E.; Mahmood, A.; Schubert, J.; Pickartz, G.; Lee, T. K. Ferroelectric properties of compressively strained epitaxial SrTiO3 films on sapphire Journal of the European Ceramic Society, 27 (2007), 2899 - 2902 Wrobel, G.; Hller, M.*; Ingebrandt, S.; Dieluweit, S.; Sommerhage, F.; Bochem, H. P.; Offenhusser, A. Transmission electron microscopy study of the cell-sensor interface Journal of the Royal Society Interface, 10 (2007), 1094 - 1098 Wrobel, G.; Zhang, Y.; Krause, H.-J.; Wolters, N.; Sommerhage, F.; Offenhusser, A. Influence of the first amplifier stage in MEA systems on extracellular signal shapes Biosensors and Bioelectronics, 22 (2007), 1092 - 1096 Wu, Y. H.; Panaitov, G.; Zhang, Y.; Klein, N. Design and fabrication of in-plane resonant microcantilevers Microelectronics Journal, 39 (2007), 44 - 48 Yeung, C. K.*; Sommerhage, F.; Wrobel, G.; Offenhusser, A.; Chan, M.*; Ingebrandt, S. Drug Profiling Using Planar Microelectrode Arrays Analytical and Bioanalytical Chemistry, 387 (2007), 2673 - 2680 Zhang, Y.; Qiu, L.; Krause, H.-J.; Hartwig, S.*; Burghoff, M.*; Trahms, L.* Liquid state nuclear magnetic resonance at low fields using a nitrogen cooled superconducting quantum interference device Applied Physics Letters, 90 (2007), 182503

2008
Abouzar, M. H.; Poghossian, A.; Razavi, A.; Besmehn, A.; Bijnens, N.*; Williams, O. A.*; Haenen, K.*; Wagner, P.*; Schning, M. J. Penicillin detection with nanocrystalline-diamond field-effect sensor Physica Status Solidi A, 205 (2008) 9, 2141 - 2145 Barannik, A. A.*; Bunyaev, S. A.*; Cherpak, N. T.*; Vitusevich, S. A. Quasioptical Sapphire resonators in the form of a truncated cone Journal of Lightwave Technology, 26 (2008) 17, 3118 - 3123 Beigmohamadi, M.*; Niyamakom, P.*; Farahzadi, A.*; Effertz, C.*; Kremers, S.*; Brggemann, D.; Wuttig, M.* Structure and morphology of perylene films grown on different substrates Journal of Applied Physics, 104 (2008) 1 167

Belyaev, A. E.*; Raicheva, V.G.*; Kurakin, A. M.; Klein, N.; Vitusevich, S. A. Investigation of spin-orbit interaction in AlGaN/GaN heterostructures with large electron density Physical Review B, 77 (2008) 3, 035311 Bettaeib, L.*; Kokabi, H.*; Poloujadoff, M.*; Sentz, A.*; Krause, H.-J. Non destructive testing (NDT) with high-Tc rf SQUIDs Journal of Physics : Conference Series, 97 (2008), 012263 Borstlap, D.; Schubert, J.; Zander, W.; Offenhusser, A.; Ingebrandt, S. High-k dielectric layers for bioelectronic applications IEICE Transactions on Electronics, E91-C (2008) 12, 1894 - 1898 Crisan, A.*; Wrdenweber, R.; Hollmann, E.; Kutzner, R.; Button, T. W.*; Abell, J. S.* Thermally-induced self-assembling nanotechnology of gold nano-dots on CeO2-buffered sapphire for superconducting films Journal of Optoelectronics and Advanced Materials, 10 (2008), 1370 - 1373 Gilioli, E.; Baldini, M; Bindi, M.; Bissoli, F.; Calestani, D.; Pattini, F.; Rampino, S.; Rocca, M.; Zannella, S.; Wrdenweber, R. Pulsed Electron Deposition (PED) of Single Buffer Layer for 'low-cost' YBCO Coated Conductors Journal of Physics : Conference Series, 97 (2008), 012197 Gordan, O. D.*; Persson, B. N. J.*; Cesa, C. M.; Mayer, D.; Hoffmann, B.; Dieluweit, S.; Merkel, R. On Pattern Transfer in Replica Molding Langmuir, 24 (2008), 6636 - 6639 Klushin, A. M.; He, M.; Levitchev, M. Yu.; Kurin, V. V.; Klein, N. Optimization of the coupling of mm wave power to arrays of high-Tc Josephson junctions Journal of Physics : Conference Series, 97 (2008), 012268 Kurakin, A.; Vitusevich, S.; Danylyuk, S.; Hardtdegen, H.; Klein, N.; Bougrioua, Z.*; Danilchenko, B. A.*; Konakova, R. V.*; Belyaev, A. E.* Mechanism of mobility increase of the two-dimensional electron gas in AlGaN/GaN heterostructures under small dose gamma irradiation Journal of Applied Physics, 103 (2008), 083707 Kuzel, P.*; Kadlec, C.*; Kadlec, F.*; Schubert, J.; Panaitov, G. Field-induced soft mode hardening in SrTi03/DySc03 multilayers Applied Physics Letters, 93 (2008), 052910-1 - 052910-3 Lukashenko, A.*; Wrdenweber, R.; Ustinov, A. V.* Imaging of vortex flow in microstructured high-Tc films by laser scanning microscope Physica C, 468 (2008), 552 - 556 Meier, M.; Nauenheim, C.; Gilles, S.; Mayer, D.; Kgeler, C.; Waser, R. Nanoimprint for future non-volatile memory and logic devices Microelectronic Engineering, 85 (2008), 870 - 872 Qiu, L. Q.; Zhang, Y.; Krause, H.-J.; Braginski, A. I. SQUID-detected NMR in Earth's magnetic field Journal of Physics : Conference Series, 97 (2008), 012026 Reska, A.; Gasteier, P.*; Schulte, P.; Mller, M.*; Offenhusser, A.; Groll, J.* Ultrathin Coatings with Change in Reactivity over Time Enable Functional In Vitro Networks of Insect Neurons Advanced Materials, 20 (2008), 2751 - 2755 Schrper, F.; Brggemann, D.; Mourzina, Y.; Wolfrum, B.; Offenhusser, A.; Mayer, D. Analyzing the electroactive surface of gold nanopillars by electrochemical methods for electrode miniaturization Electrochimica Acta, 53 (2008), 6265 - 6273 Sommerhage, F.; Helpenstein, R.; Rauf, A.; Wrobel, G.; Offenhusser, A.; Ingebrandt, S. Membrane allocation profiling: A method to characterize three-dimensional cell shape and attachment based on surface reconstruction Biomaterials, 29 (2008), 3927 3935 Vitusevich, S. A.; Kurakin, A. M.; Klein, N.; Petrychuk, M. V.*; Naumov, A. V.*; Belyaev, A. E.* AlGaN/GaN High Electron Mobility Transistor Structures: Self-Heating Effect and Performance Degradation IEEE Transactions on Device and Materials Reliability, 8 (2008) 3, 543 - 548 Vitusevich, S. A.; Kurakin, A. M.; Konakova, R. V.*; Belyaev, A. E.*; Klein, N. Improvement of interface properties of AlGaN/GaN heterostructures under gamma-radiation Applied Surface Science, 255 (2008) 3, 784 - 786 168

Institute of Bio- and Nanosystems Biomechanics (IBN-4) 2007

Beltramo, G.; Ibach, H.; Mller, J. E.; Giesen, M. A Novel Approach to Measure the Step Line Tension and the Step Dipole Moment on Vicinal Au(001) Electrodes Surface Science, 601 (2007), 1876 - 1885 Cesa, C. M.; Kirchgener, N.; Mayer, D.; Schwarz, U. S.*; Hoffmann, B.; Merkel, R. Micropatterned silicon elastomer for high resolution analysis of cell force patterns Review of Scientific Instruments, 78 (2007) 3, 034301 Hartwig, B.*; Borm, B.; Schneider, H.*; Arin, M. J.*; Kirfel, G.*; Herzog, V.* Laminin-5-deficient human keratinocytes: Defective adhesion results in a saltatory and inefficient mode of migration Experimental Cell Research, 313 (2007) 8, 1575 - 1587 Ikonomov, J.*; Starbova, K.*; Giesen, M. Measurements of Step and Kink Energies and of the Step Edge Stiffness from Island Studies on Pt(111) Physical Review B, 75 (2007), 245411-1 - 24541-8 Ikonomov, J.; Starbova, K.; Giesen, M. Island coalescence and diffusion along kinked steps on Cu(001): Evidence for a large kink Ehrlich-Schwoebel barrier Surface Science, 601 (2007) 5, 1403 - 1408 Limozin, L.*; Sengupta, K. Modulation of Vesicle Adhesion and Spreading Kinetics by Hyaluronan Cushions Biophysical Journal, 93 (2007), 3300 - 3313 Merkel, R.; Kirchgener, N.; Cesa, C. M.; Hoffmann, B. Cell Force Microscopy on Elastic Layers of Finite Thickness Biophysical Journal, 93 (2007), 3314 - 3323 Pichardo-Pedrero, E.*; Beltramo, G.; Giesen, M. Electrochemical annealing and its relevance inmetal electroplating: An atomistic view Applied Physics A, 87 (2007), 461 - 467 Pichardo-Pedrero, E.; Giesen, M. Comparative STM Studies on island equilibrium shapes, shape fluctuations and island coalescence on Au(001) electrodes in chloric and sulfuric acid solutions Electrochimica Acta, 52 (2007) 18, 5659 - 5668 Schaap, I.A.T.*; Hoffmann, B.; Carrasco, C.*; Merkel, R.; Schmidt, C. F.* Tau protein binding forms a 1 nm thick layer along protofilaments without affecting the radial elasticity of microtubules Journal of Structural Biology, 158 (2007), 282 - 292 Semmrich, C.*; Storz, T.*; Glaser, J.*; Merkel, R.; Bausch, A. R.*; Kroy, K.* Glass transition and rheological redundancy in F-actin solutions (from the cover) Proceedings of the National Academy of Sciences of the United States of America, 104 (2007), 20199 - 20203 Wrobel, G.; Hller, M.*; Ingebrandt, S.; Dieluweit, S.; Sommerhage, F.; Bochem, H. P.; Offenhusser, A. Transmission electron microscopy study of the cell-sensor interface Journal of the Royal Society Interface, 10 (2007), 1094 - 1098

2008
Beltramo, G. L.; Ibach, H.; Linke, U.; Giesen, M. Determination of the step dipole moment and the step line tension on Ag(001) electrodes Electrochimica Acta, 53 (2008), 6818 - 6823 169

Biehl, R.; Hoffmann, B.; Monkenbusch, M.; Falus, P.*; Prost, S.*; Merkel, R.; Richter, D. Direct Observation of Correlated Interdomain Motion in Alcohol Dehydrogenase Physical Review Letters, 101 (2008), 138102-1 - 138102-4 Gordan, O. D.*; Persson, B. N. J.*; Cesa, C. M.; Mayer, D.; Hoffmann, B.; Dieluweit, S.; Merkel, R. On Pattern Transfer in Replica Molding Langmuir, 24 (2008), 6636 - 6639 Kajzar, A.*; Cesa, C. M.*; Kirchgener, N.; Hoffmann, B.; Merkel, R. Toward Physiological Conditions for Cell Analyses: Forces of Heart Muscle Cells Suspended between Elastic Micropillars Biophysical Journal, 93 (2008), 1854 - 1866 Scheijen, F. J. E.*; Beltramo, G. L.; Hoeppener, S.*; Housmans, T. H. M.*; Koper, M. T. M.* The electrooxidation of small organic molecules on platinum nanoparticles supported on gold: influence of platinum deposition procedure Journal of Solid State Electrochemistry, 12 (2008), 483 - 495 Smith, A.*; Sengupta, K.; Goennewein, S.*; Seifert, U.*; Sackmann, E.* Force-induced growth of adhesion domains is controlled by receptor mobility Proceedings of the National Academy of Sciences of the United States of America, 105 (2008), 6906 - 6911 Tschersich, K. G.; Fleischhauer, J.P.; Schuler, H.* Design and characterization of a thermal hydrogen atom source Journal of Applied Physics, 104 (2008), 034908

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Selected results 2007/2008