SPR

CHAPTER 5
Kinetic and Thermodynamic Analysis of LigandReceptor Interactions: SPR Applications in Drug Development
NICO J. DE MOL AND MARCEL J.E. FISCHER
Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands
5.1 Introduction
Increasing evidence can be found that describing receptor ligand interactions in terms of a lock-and-key model is no longer adequate. Receptors can be regarded as part of a molecular machinery, in which ligand binding forms a trigger to activate or deactivate the machinery. According to this view, it is no longer sucient to know how the key ts into the lock, but we should also nd out the mechanism with which the key opens and closes the lock. In other words, in drug design we would be interested not only in the anity of the ligand for the receptor, but also in the changes of a biological receptor molecule when it forms a complex with a ligand. Such changes may involve conformational adaptation, changes in solvation (i.e. ordering of water molecules) and changes in molecular exibility. Kinetics is a rather underdeveloped aspect of ligandreceptor interactions. It is readily conceivable that in some cases, such as in dynamic regulation of signal transduction processes, kinetic control prevails rather than anity control. Rapid onset of formation and an optimum lifetime of the complex can be ne tuned by appropriate association and dissociation kinetics. Explicit references to the biological signicance of binding kinetics are rather scarce; some examples are given by Schreiber [1]. Other examples include the serial triggering
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of T-cell receptors [2] and the activation of the epidermal growth factor receptor ErbB-1 [3]. Elucidation of the molecular architecture, using especially X-ray and NMR techniques has been of crucial importance for understanding how a ligand protein or proteinprotein interaction functions in the molecular machinery. However, for a more complete understanding of the dynamic processes underlying receptor activation, kinetic and thermodynamic studies of ligand receptor interactions are needed. It is increasingly acknowledged that, to fully appreciate relevant molecular properties of potential drug candidates in a drug design process, there is a need for thermodynamic and kinetic studies [48]. Traditionally, vant Ho analysis has been used for thermodynamic studies. More recently, the use of sensitive calorimetric techniques in drug research is emerging [9,10]. Stopped-ow has been the method of choice to study kinetics of molecular interactions. With SPR one now can derive kinetic and thermodynamic parameters from a single set of experiments. SPR allows to follow the mass change on the sensor surface in real time, yielding anity and kinetic data. Thermodynamic and kinetic parameters can be derived from a series of experiments in a temperature range. The combination of kinetic and thermodynamic information from well-designed SPR experiments is unique and oers an added value compared with separate techniques for kinetic and thermodynamic information: it allows even a full transition state analysis of the binding process from a single data set. In this chapter, we describe examples of thermodynamic and kinetic analysis of biomolecular interactions using SPR-based approaches that we have developed in recent years. These examples apply mainly to peptides, interacting monovalently or bivalently with important signal transduction proteins, containing Src Homology 2 (SH2) and SH3 domains. These signal transduction proteins are attractive targets for drug design. The underlying investigations are aimed at validation of the SPR-based approach, at gaining insight into the mechanism of the binding process and nally at using this insight in ligand design. To be able to exploit SPR fully, a few initial problems had to be solved. Part of these problems originated from the fact that in our investigations cuvettebased SPR instruments were used. As discussed in Chapters 3 and 4, in owbased SPR instruments (e.g. Biacore), a continuous ow of the sample enhances diusion of analyte towards the sensor surface. In cuvette-based instruments, the hydrodynamic properties are controlled by constant agitation of the bulk solution in contact with the sensor surface. The cuvette-based design oers the following advantages: (1) open architecture allowing manual interventions during a run and (2) long association times without extensive consumption of often precious biological material. A disadvantage might be that during the binding process the concentration of unbound analyte in the cuvette is not constant. In this chapter, a correction for this eect is described. Another complication associated with the cuvette design is that in the dissociation phase the analyte released is not removed from the bulk solution. This problem has been solved by adding competing ligand to prevent rebinding of released analyte during the dissociation phase.
Kinetic and Thermodynamic Analysis of LigandReceptor Interactions
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When the association rates are high compared with the diusion in the bulk solution, mass transport limitation (MTL) occurs.1 Association and dissociation are aected by MTL to the same extent. We describe a simple method to estimate the extent of MTL. As MTL can be easily included in a simple kinetic model, the experimental association curves can be analyzed. Another problem, not related to instrumental design, is that in principle the anity of the analyte for the immobilized ligand at the sensor surface, as obtained in a direct SPR assay, is not necessarily identical with that in solution. A method is described to obtain thermodynamic binding constants for the interaction in solution, using competition experiments with a concentration range of the ligand of interest. Using this approach, several ligands can be studied using the same sensor surface. We should emphasize that in this chapter the focus is not so much on theory, but rather on application. We would like to give the reader practical tools to obtain reliable kinetic and thermodynamic parameters on the binding processes. In order to achieve this, we need to provide the corresponding conceptual and theoretical background. Following the outlined approach, reliable kinetic and thermodynamic parameters can be obtained, which can greatly increase our knowledge of binding processes. Later in this chapter we show examples of how kinetic and thermodynamic analysis of interactions using SPR can support chemical biology studies in general and rational structure-based drug design in particular.
5.2 Anity and Kinetics of a Transport-limited Bimolecular2 Interaction at the Sensor Surface
In a standard SPR assay, one of the interacting partners (the ligand) is immobilized on the sensor surface. The other component (the analyte) is added in the solution, in our case in a cuvette. In our experiments, the ligand is usually a peptide provided with a linker, to increase the distance between the binding epitope and the matrix on the sensor surface, avoiding steric hindrance between the bound analyte and the sensor matrix (see Figure 5.1). The linker is also provided with a free NH2 terminus, for covalent coupling to the sensor surface using EDC/NHS chemistry.3 This system guarantees a homogeneous surface by uniform coupling of the ligand through the NH2 group. The analyte is a protein with generally a much higher molecular weight than the ligand. This increases the sensitivity of the assay, as the change in SPR angle is proportional to the amount of bound mass. Hydrogel SPR sensor chips are used, containing carboxymethylated dextran chains on a 50 nm gold surface (Figure 5.1), either from Biacore (Uppsala, Sweden) or Xantec (Mu nster, Germany). Negatively charged ligands, e.g. peptides
1
For a more detailed treatment of mass transport limitation and diusion, see Chapter 4, Section 4.2.1. 2 A bimolecular interaction is a biomolecular interaction of only one analyte (A) which binds with one ligand (B) to form complex (AB). 3 For further details, see Chapter 7.
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Figure 5.1
Schematic view showing (a) the SPR sensor matrix existing of dextran chains with carboxymethyl groups on a gold surface (50 nm) before coupling, (b) immobilization of the ligand after coupling and (c) binding of analyte to the surface.
containing phosphotyrosines (pY), are more dicult to immobilize, due to lack of preconcentration at the sensor matrix, caused by electrostatic repulsion between the negatively charged peptide and the negatively charged carboxyl groups on the dextran chains. In such cases 1 mol l1 NaCl is added to the coupling buer to diminish electrostatic repulsion [11]. Our SPR instruments (IBIS and Autolab ESPRIT) have two cuvette cells: a sample cell and a reference cell. The two cells are treated in an identical way, the only dierence being that only the sample cell contains immobilized ligands. The net SPR signal (the signal in the reference cell subtracted from the signal in the sample cell) is used for further analysis. Subtraction of the reference signal allows correction for bulk eects due to addition of the analyte, for transient temperature eects and for non-specic binding which occurs incidentally. In a series of experiments at dierent analyte concentrations, the anity of the analyte for the immobilized ligand can be assayed in several ways. The method preferred by us is non-linear tting of the SPR signal at equilibrium with a Langmuir binding isotherm. Alternative methods are based on the kinetics of the interaction. These methods for determining the anity of the analyte will be described in the following sections.
5.2.1 Anity Constants Derived from Equilibrium SPR Signals

For a simple bimolecular interaction with molecules A and B forming the complex AB, the equilibrium association constant KA and dissociation constant KD are given by eqs. (5.1a) and (5.1b): KA AB ; with KA in l mol1 AB 5:1a
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KD
AB 1 ; with KD in mol l1 AB KA
5:1b
where the brackets [A], etc., indicate concentration of the molecules. In a welldesigned anity experiment, several analyte concentrations are used, which should be in a range around the KD value. In SPR experiments, [AB] and [B] are not approached as concentrations in solution, but as amounts at the surface expressed as SPR signal. The amount of complex AB is proportional to the shift in SPR angle [expressed in millidegrees (m1) or so-called response units (RU)]. A conversion factor can be calculated for SPR response to concentrations in the volume of the 100 nm dextran layer at the sensor surface (see, e.g., Box 5.1). The shift in SPR angle is recorded as function of time in a sensorgram. In Figure 5.2, an example of sensorgrams, based on the net SPR signal (Rsample cell Rreference cell), at dierent analyte concentrations is shown. Using the kinetic evaluation software supplied with SPR instruments, the shift in SPR angle at equilibrium (Req) is readily determined (see Section 5.2.2.1). Frequently, the data are represented in a Scatchard plot (Req/[analyte] vs. Req) as a straight line. However, large errors can occur in Scatchard plots, especially at low concentrations, with small amounts of binding [13], therefore we prefer non-linear regression using the Langmuir binding isotherm [eq. (5.2)], in which [A] is the free analyte concentration and Bmax is the maximum binding capacity in m1, when all binding sites on the sensor surface are occupied. A Req 5:2 Bmax A KD Examples of plots with ts according to the Langmuir binding isotherm are shown in Figure 5.3.
Figure 5.2
Sensorgrams (net signal) of binding of v-Src SH2 protein to immobilized EPQpYEEIPIYL-peptide. Start of dissociation is indicated by the arrow. v-Src SH2 concentrations form top to bottom: 500, 333, 208, 125, 83.3 and 62.5 nmol l1. For further details, see ref. [12].
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Figure 5.3
SPR signal at equilibrium as function of analyte concentration. The lines are the ts with the Langmuir binding isotherm [eq. (5.2)]. Data without depletion correction, open circles; with depletion correction (see Section 5.2.1.1), closed circles. (A) Binding of v-Src SH2 domain (conditions as in Figure 5.2). (B) Binding of Syk kinase tandem SH2 domain. For further details on this interaction, see ref. [14].
5.2.1.1
Correction for Depletion of Free Analyte Concentration in the Cuvette
In a cuvette, the free analyte concentration decreases due to binding to the sensor. This section describes how depletion of analyte can be quantied and corrected for. The change in SPR angle (in m1) due to a binding process is directly related to the amount4 of bound material per mm2. Under equilibrium conditions the amount of bound analyte is proportional to Req (in m1) and the surface of the sensor S (in mm2) in contact with the bulk solution. To relate the amount of bound analyte to a decrease in the free analyte concentration, the molecular weight (MW) of the analyte and the volume of the bulk solution (Vbulk, in liters) must be known. The depletion correction can be calculated using eq. (5.3). Afree A0 Req S 109 122 MW Vbulk 5:3
Here [A]0 is the initially added analyte concentration in the bulk and [A]free is the corrected analyte concentration, both in nmol l1. Two examples of depletion correction are presented in Figure 5.3 in (A) for v-Src SH2 with molecular weight 12 300 Da and in (B) Syk kinase tandem SH2
For the IBIS and Autolab ESPRIT instruments used by us, an SPR signal of 122 m1 corresponds to 1 ng mm2 at 25 1C.
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(Syk tSH2) with MW is 29 800 Da. With eq. (5.3), using values for S (6 mm2) and Vbulk (35 ml, the applied volume), the depletion correction is calculated as 0.114 nmol l1/m1 for v-Src SH2 protein and 0.0472 nmol l1/m1 for Syk tSH2 protein. It is obvious from Figure 5.3 that for Syk tSH2 the depletion correction has a larger eect: without correction KD is 8.7 nmol l1 and with correction KD is 5.9 nmol l1. Although the correction factor for v-Src protein is larger due to the lower molecular weight, the depletion correction has only a limited eect in this case: KD goes from 294 nmol l1 without correction to 252 nmol l1 with correction. For Syk tSH2 the eect of correction on KD is much larger. Owing to the high anity, the Syk tSH2 concentration used in the assay is very low (Figure 5.3). Depletion caused by binding to the sensor surface has a large eect at low concentrations. Another factor with direct eect on depletion correction is the binding capacity Bmax of the sensor surface, as it is directly proportional to Req [see eq. (5.2)]. To minimize depletion and the need for correction, a low binding capacity is advised. In general, a value of Bmax of 100 m1 (or 500 RU in Biacore systems) is more than sucient for reliable assays.5 In equilibrium anity assays using, e.g., the Langmuir binding isotherm, the depletion correction can be readily calculated by entering the proper numbers in eq. (5.3) and by using a spreadsheet, the correction can be automatically calculated for every data point. Problems may arise when the sensorgrams are used for kinetic analysis. If the depletion correction is large, the free analyte concentration will substantially diminish during the association phase and second order kinetics might apply [15]. In our experience, as long as the depletion correction is below 10% of the total analyte concentration, rstorder kinetics can be safely used [11]. From eq. (5.3) it can be concluded that if Bmax is below 100 m1, for medium strong interactions (KD E 100 nmol l1) and analyte molecular weights higher than 10 kDa, depletion corrections will be smaller than 10%. In kinetic analysis of high-anity interactions as in the case of Syk tSH2 (see Section 5.4.1), one should be aware of the occurrence of second-order kinetics. In these systems, reliable kinetic analysis is possible based on rst-order association kinetics, on surfaces with low Bmax (B50 m1) and only at higher concentrations, such that depletion correction remains below B10%.
5.2.2 Anity Constants and Rate Constants Derived from Kinetic Analysis
In the previous section we focused on equilibrium anity assays based on Req. Alternatives are oered by kinetic analysis based on the shape of the sensorgrams, which can be useful when the association rate is slow. Especially in owbased SPR instruments lengthy association times to reach equilibrium may cause problems due to large analyte consumption.
5
Or even lower, depending on the sensitivity of the instrument.
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5.2.2.1
kobs Kinetic Analysis
Assuming a simple bimolecular interaction with analyte A interacting with immobilized ligand B, forming the complex AB at the sensor surface, ideally the SPR signal vs. time (Rt) is given by eq. (5.4) [16]. kon ABmax 1 ekon Akoff t kon A koff
Rt
5:4
Here kon and ko are the association and dissociation rate constants for formation and dissociation of the complex AB, respectively. Note that Rt is proportional to the amount of complex AB. A new parameter kobs6 is dened as kobs kon[A] + ko. Using software that is generally supplied with SPR instruments, a t of the curve of Rt vs. time yields kobs. When kobs is plotted vs. [A], kon can be obtained from the slope of the curve and ko from the intercept. The anity can be calculated as KA kon/ko or KD ko/kon. In Figure 5.4, examples of kobs analysis are shown, using the data sets of Figure 5.3. The parameters of the kobs analysis for v-Src SH2 are included in Table 5.1. Although the plots are linear, as expected from theory, the results deviate from the equilibrium analysis. Now for v-Src SH2 a KD value of 11.9 nmol l1 is found, which is almost two orders higher than obtained from the Langmuir binding isotherm. For Syk tSH2 no anity could be determined using this analysis because the intercept is slightly below zero. The reason for these deviations is that these interactions are severely aected by mass transport limitation (MTL), as appears in Sections 5.2.2.2 and 5.3. Under such conditions, eq. (5.4), which forms the base for this analysis, no longer holds. Schuck and Minton [17] showed with theoretical data how MTL inuences the kobs vs. [A] plot. Further examples of how MTL aects the outcome of kobs analysis can be found in the literature [17,18]. As shown above, a straight kobs plot by no means indicates that reliable kinetic data can be derived. Unfortunately, a number of examples of erroneous interpretations of kinetic data can be found in the literature, especially using kobs-analysis or closely related methods. To avoid this pitfall, one should be absolutely sure that MTL is not involved. A number of simple selfconsistency tests, e.g. comparing data from equilibrium and kinetic analyses, should be performed before interpreting such kinetic analysis [20]. A simple experiment to test whether MTL is involved is to measure the eect of addition of binding ligand during analyte dissociation to prevent rebinding (see Section 5.3.2.2). The kobs analysis presented in the previous section has severe shortcomings as the outcome is very sensitive to MTL. In the following, an alternative model is oered which also includes MTL. This approach is based on analysis of sensorgrams and curve tting according to predened binding models.
6
In earlier publications regarding kinetic evaluations [16], this parameter is denoted ks.
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Figure 5.4
kobs plots for binding of v-Src SH2 (closed circles) and Syk tSH2 (open circles) to immobilized ligands. Analysis of datasets presented in Figure 5.3.
Table 5.1
Kinetic and anity parameters for v-Src SH2 protein binding to immobilized EPQpYEEIPIYL-peptide (experimental data shown in Figure 5.2), as derived with dierent approaches (see text).
CLAMP global analysis Binding isotherm 252 13 323 8 kobs analysis 11.9a 9.2 (0.3) 104 1.1 (0.8) 103 Model 1 308a 363 3 3.35 104 0.01 4.19 Model 2 250a 323 7.99 106 2b 6.3 106 2.00
Method parameter KD (nmol l1) Bmax (m1) kon (l mol1 s1) ko (s1) Lm (m s1)c Res Ssqd
a b c
Calculated from ko/kon. Experimental value from dissociation in the presence of peptide to prevent rebinding. Calculated from ktr with conversion factor (see Box 5.1). d Residual sum of squares [19], indicates quality of the t.
5.2.2.2
Global Kinetic Analysis with a Simple Bimolecular Binding Model
The real-time information on the mass changes resulting from the interaction can be used to study various binding models, also including MTL. In this
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1: Bimolecular model A+ B
k on k off
2: Bimolecular model + transport step
AB
A0
ktr k tr
A
k on k off
A+ B
AB
Scheme 5.1
Binding models for a simple bimolecular reaction (1) and a bimolecular reaction including a transport step of analyte from the bulk to the sensor surface (2).
section, the emphasis is on the rate constants of a simple, transport-limited bimolecular reaction, more complicated binding models are presented in Section 5.4. In this chapter, the kinetic analysis is treated using basic chemical kinetics concepts applied to experimental SPR data. In Chapter 4, kinetics is treated with concepts based on physical solute absorption to surfaces.7 In general, dierential rate equations for species binding to the sensor surface can be derived from a binding model. Experimental sensorgrams can be tted to a model and one can analyze how far the experimental data agree with the model. Furthermore, parameters such as rate constants and maximum binding capacity can be derived from the ts. In a global analysis such t procedures are applied to several curves obtained at dierent analyte concentrations simultaneously, using the same t parameters. To explain the procedure we use a simple bimolecular binding model with and without mass transport step (see Scheme 5.1). For model 1, the following dierential rate equations can be derived: Association : dAB kon AB koff AB dt dAB koff AB dt 5:5a 5:5b
Dissociation :
The analytical solution for the association phase is eq. (5.4) and for dissociation it is eq. (5.6), where [AB] is directly proportional to Rt, the net SPR signal at time t, and Req the net SPR signal at dissociation time zero. Equation (5.6) describes rst-order exponential decay from which ko can be obtained. Dissociation : Rt Req ekoff t 5:6
Model 2 is somewhat more complicated: again in the association phase the time dependence of [AB] is given by the dierential rate equation [eq. (5.5a)], but now also the time dependence of diusion of analyte from the bulk ([A]0) to the sensor surface ([A]) has to be taken into account. The accompanying rate equations are given in eqs. (5.7a) and (5.7b). dA0 ktr A0 ktr A dt
7
5:7a
Remark: this is also the reason why terminology, symbols, etc., dier in Chapters 4 and 5.
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Figure 5.5
Global analysis using the program CLAMP of the association phase of binding of v-Src SH2 protein to a pY-containing peptide (data set as in Figure 5.2). Black lines, experimental curves, red lines, tted curves. (A) Fit with bimolecular model 1, all t parameters are left free. (B) Simulation of model 1 with xed parameters for Bmax (320 m1), kon (8 106 l mol1 s1) and ko (2 s1). (C) Fit with transport model 2, with ko xed on experimental value. See text for further details.
dA ktr A0 ktr A kon AB koff AB dt
5:7b
Several programs can be used for solving such dierential equations by numerical integration. We used the program CLAMP developed for tting experimental sensorgrams [19].8 Consistency of the ts is greatly improved by tting several curves for dierent analyte concentrations simultaneously with the same kinetic parameters in a so-called global analysis. Examples of global kinetic analysis with CLAMP are shown in Figure 5.5, with the data set of Figure 5.2. To emphasize the kinetic phase, only a relatively short association time interval was allowed. The quality of the ts compared with the experimental data is indicated by the residual sum of squares (res Ssq) parameter [19]. For models 1 and 2 this is rather similar (Table 5.1). However, the initial linear phase observed for the higher concentrations is not very well tted with model 1, and the t returns a ko value of 0.01 s1. This linear phase is indicative for MTL [21]. From experiments in the presence of competing peptide to prevent rebinding of protein during the dissociation phase (see Section 5.3.2.2), a much higher experimental value of 2 s1 for ko is obtained. Therefore, we conclude that this model does not yield a satisfactory description of the kinetic parameters. The experimental values of KD and Bmax are known from the binding isotherm (Figure 5.3), ko is known from dissociation experiments and kon can be calculated from ko/KD. Therefore, all parameters in model 1 are known, allowing simulation of the sensorgrams based on model 1 (Figure 5.5B). This simulation demonstrates that in practice the association phase proceeds much slower than expected for the high on-rate of 8 106 l mol1 s1. This
8
Currently, the features of CLAMP are included in a more extended biosensor data analysis tool named Scrubber2 from the results of David Myszka (see http://www.cores.utah.edu/interaction/ software.html).
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underscores MTL: due to the high on-rate, diusion of analyte to the sensor surface is much slower and becomes rate limiting. In model 2, a step for transport of analyte from the bulk to the sensor surface is included. This model, using the xed experimental ko value of 2 s1, gives an excellent t to the experimental data. The diusion of analyte to and from the sensor surface is assumed to be equal and is characterized by the rate constant ktr. From eq. (5.7b) it follows that the units of ktr obtained from the CLAMP t are m1 s1 l mol1, as [B] and [AB] are in m1 and the tted curves are SPR signal (m1) vs. time (s). The value of ktr in m1 s1 l mol1 can be converted to the mass transport coecient [21] (Lm) in m s1 (see Box 5.1). For v-Src SH2 protein (MW 12.3 kDa) this conversion factor is 6.66 1013; applying this conversion yields Lm in m s1. In Table 5.1, the anity and kinetic parameters derived from global analysis of the dataset of Figure 5.2, using models 1 and 2, are included. The results illustrate once more that in this severely transport-limited system the outcome of kobs analysis is not reliable. The anity from MTL model 2 agrees perfectly with that from equilibrium analysis using the binding isotherm (Table 5.1). This is not surprising, as in a considerable part of the tted curves the signal is at equilibrium (see Figure 5.5C). In model 2, the use of an experimental value for ko is crucial for the outcome. In general, it helps to use in the ts xed experimental values for, e.g., ko and Bmax.
Box 5.1 Conversion of ktr (in m1 s1 l mol1) into the mass transfer coecient Lm (in m s1)
To calculate Lm two conversions have to be applied: (1) from m1 to mol m2 and (2) from l mol1 to m3 mol1. 1. The SPR signal in m1 corresponds to a xed amount of material/surface unit. For the IBIS and Autolab ESPRIT instruments used in these studies, 122 m1 corresponds to 1 ng mm2 or 103 g m2. Taking into account the molecular 103 2 weight (MW) of the analyte, 1 m1 corresponds to 122 MW mol m . 1 3 1 3 3 1 2. 1 l mol is 1 dm mol ; this corresponds to 10 m mol . Combining 1 and 106 2, the conversion factor from m1 s1 l mol1 to m s1 is 122 MW. For v-Src SH2 protein with an MW of 12.3 kDa, the conversion factor is 6.66 1013. From the t with model 2, ktr is found to be 9.49 106 m1 s1 l mol1. Applying the conversion factor, this corresponds to Lm 6.3 106 m s1. In Biacore instruments, the SPR signal is expressed in response units (RU); 1 RU corresponds to 1 pg mm2. As 122 m1 corresponds to 1 ng mm2 (see above), 1000 RU corresponds to 122 m1, and 1 m1 is 8.2 RU. In this chapter, calculations are based on m1. By using the conversion factor of 8.2, these calculations can be adapted for RU-values.
It is surprising that the experimental data can be described by such relatively simple models. For example, usually not all binding sites are equal: within the
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dextran layer of the sensor the binding sites more close to the gold surface have a higher intrinsic contribution to the SPR signal, due to the exponentially decaying evanescent eld (Chapter 2). Furthermore, especially on high binding capacity surfaces approaching saturation of binding, heterogeneity of binding sites is expected. The global kinetic analysis presented here is attractive because it yields thermodynamic and kinetic parameters. However, one should be careful in the interpretation of kinetic parameters as in the t procedure these can be mutually correlated [22] and in more complicated binding models the separate steps may not be completely kinetically resolved, as described in Section 5.4.
5.3 Detecting Mass Transport Limitation: A Practical Approach

Kinetic and anity analysis with simple models can lead to large errors when MTL is unaccounted for, as shown in Section 5.2. Therefore, it is necessary to detect MTL. In this section, practical methods are described to nd transport limitation.9 Furthermore, we describe here two approaches to obtain true o-rates10 from severely MTL-aected dissociation phases.
5.3.1 Eect of Viscosity Change on the Association Phase

The essence of MTL is that the on-rate is high and diusion of analyte from the bulk phase to the biosensor (and partly in the biosensor dextran layer [23]) becomes rate limiting. Viscosity changes of the bulk solution will aect diusion of the analyte and this should be visible in the sensorgrams of an MTL-controlled interaction. We performed experiments with increasing amounts of glycerol to increase the viscosity. In Figure 5.6, the eect of glycerol on the association of the GST fusion protein of the Lck SH2 domain to immobilized pY-peptide is shown. As expected, increasing the viscosity slows down the association. No eect of glycerol in the applied amounts on the anity was observed (equilibrium signal not aected). Kinetic analysis of data sets obtained with a range of glycerol concentrations, using model 2 (Scheme 5.1), yields a series of ktr values and Lm transport coecients. For ow cells, the ux to the sensor surface due to mass transfer (i.e. Lm) was derived to be proportional to D2/3 [24]. The diusion coecient D is reciprocally related to the viscosity Z, according to the Stokes Einstein equation, and therefore Lm should be proportional to Z2/3. From Figure 5.7, it appears that a plot of Lm vs. Z2/3 yields a linear relation as predicted for ow systems. For a cuvette instrument, the hydrodynamics might be dierent, as the bulk solution is subject to continuous agitation. Actually, with this data range it is not possible to discern the ow model from other models, as a plot of Lm vs. Z also has an excellent linear correlation.
9 10
For a more basic treatment of mass transport phenomena, see Chapter 4. From theoretical considerations by Schuck and Minton [17], it follows that MTL aects association and dissociation to the same extent.
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Figure 5.6
Eect of glycerol on the association phase of 30 nmol l1 Lck SH2 GST fusion protein to immobilized Ahx-EPQpYEEIPIYL-peptide. Solid line, no glycerol; dashed line, 5% glycerol; dot-dashed line, 7.5% glycerol. Reprinted from ref. [11], Copyright (2000), with permission from Elsevier.
Figure 5.7
Relation between mass transport coecient (Lm) from bulk solution to the sensor surface and viscosity (Z) as predicted for the hydrodynamics in a ow cell. Determined in a cuvette based instrument for binding of Lck SH2 GST fusion protein to immobilized EPQpYEEIPIYL-peptide in the presence of 0 to 10% glycerol.
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Attempts to correlate Lm values with molecular weight have not been successful. This is probably caused by dierences among individual sensors surfaces and the fact that Lm depends not only on diusion in the bulk solution, but also on diusion within the sensor surface dextran layer as proposed by Schuck [23].
5.3.2 Transport Limitation in the Dissociation Phase

The high on-rate compared with diusion also aects the apparent dissociation kinetics. If diusion is slow and the on-rate is high, a considerable amount of dissociated analyte will rebind before there is an opportunity to diuse away from the sensor surface into the bulk. This implies that if the association phase is transport limited, the dissociation is also transport limited. In cuvette instruments used in a static mode (i.e. released analyte is not removed from the cell), rebinding will always occur due to the equilibrium between free released analyte in the bulk and bound analyte on the sensor surface, even under conditions that transport limitation does not apply! We present two independent methods to assay true dissociation rates, which gave comparable ko values. The rst method takes rebinding into account and the second uses added competing ligands during dissociation to prevent rebinding.
5.3.2.1
Rebinding Model for Transport-limited Dissociation
If transport limitation applies, the dissociation phase for a simple bimolecular interaction on a homogenous surface will no longer be described by rst-order decay kinetics according to eq. (5.6). A high on-rate compared with diusion away from the biosensor compartment and the availability of free binding sites on the surface will increase rebinding of released analyte. Schuck and Minton [17] have developed a two-compartment model as an approximate description for the dissociation phase under ow conditions. This model is characterized by the dierential eq. (5.8). dR t koff Rt kon dt 1 ktr Bmax Rt 5:8
In this model ktr (in m1 s1 l mol1) has the same meaning as in model 2 (Scheme 5.1) and represents transport between the bulk and the sensor surface. If ktr c kon no transport limitation will occur and eq. (5.8) then changes to eq. (5.5b). (Bmax Rt) represents the amount of free binding sites and will be proportional to the amount of rebinding. An example of application of this model using the program REBIND11 is shown in Figure 5.8. Continuous wash steps were performed to remove released analyte. Initially, dissociation proceeds fast (Figure 5.8A), as at the start of the dissociation practically all binding sites are occupied and rebinding is negligible. Soon more binding sites become available, slowing dissociation due to
11
Kindly provided by Dr. Peter Schuck.
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Figure 5.8
Sensorgrams of binding of Lck SH2 GST fusion protein to immobilized EPQpYEEIPIYL peptide. (A) Solid line, dissociation without renewal of bulk solution; dashed line, dissociation with continuous wash steps to remove released protein from the cuvette. (B) Upper lines: dotted line, experimental data for dissociation with continuous wash steps; continuous line, t of the data with the program Rebind based on dierential eq. (5.8). Below: residuals of the t. Reprinted from ref. [11], Copyright (2000), with permission from Elsevier.
rebinding. Continuous renewal of the bulk solution increases the apparent dissociation rate. The dissociation phase with the wash steps is excellently matched by the model in eq. (5.8) (Figure 5.8B) with Bmax xed at the experimental value derived from the binding isotherm [eq. (5.2)]. Using the sum of squared residuals (SSR) analysis of REBIND, a large interval of 0.06 o ko o 0.95 s1 falls within 5% of the best SSR value (see Figure 5.9, lower curve). In this interval, ko appears to be strongly correlated with kon/ktr. This especially occurs if (kon/ko)(Bmax Rt) c 1 [see eq. (5.8)], which is the case under conditions of considerable transport limitation (kon and Bmax Rt are large). If a surface is used with a lower Bmax, eects of transport limitation can be somewhat diminished; however, in a severe transport-limited system, Bmax should be unrealistically low to prevent transport limitation completely (see Section 5.3.3). Introduction of experimental values for kon and ktr, as derived from global analysis of the association phase with, e.g., model 2 greatly improves the results. As can be seen in Figure 5.9, the SSR analysis indicates a discrete value for ko if kon/ktr is kept at the value from the association phase. The obtained ko-value of 0.38 s1 is close to the value found for the same interaction (0.6 s1), in the presence of large amounts of competing peptide to avoid rebinding (see Section 5.3.2.2.). The found value is also in accordance with the rapid dissociation found for other SH2 domains [25]. For several reasons, this result is impressive. First, the slow decay in the dissociation phase in Figure 5.8 in no way suggests such a high ko (see also Figure 5.10). The results conrm that ignoring transport limitation can yield
139
Figure 5.9
Sum of squared residuals (SSR) as a function of ko. Broken line, analysis with kon/ktr derived from global kinetic analysis of the association phase based on model 2. Reprinted from ref. [11], Copyright (2000), with permission from Elsevier.
several orders too low ko values, as can be seen in Table 5.1. Second, the fact that the transport parameter, ktr, derived independently from the association phase, gives consistent results in the dissociation phase is a solid experimental conrmation that both the association phase and the dissociation phase are aected to the same extent by transport limitation, as concluded from theoretical considerations [17]. In practice, it appears that dissociation curves that can be tted well with eq. (5.8) can also be well tted by a double exponential dissociation equation for two independent binding sites with each their own dissociation rate [17]. In many cases such a t will result in an artifact and the obtained rates are not meaningful. Before concluding from a double exponential t that two dierent binding sites are involved, a simple consistency check should be performed, e.g. by adding competing peptide to diminish/prevent rebinding (see Section 5.3.2.2).
5.3.2.2
Competing Ligand to Prevent Rebinding During Dissociation
As indicated in the previous section, under transport-limited conditions the dissociation phase is considerably inuenced by rebinding of released analyte. In principle, this rebinding can be prevented by adding an excess of competing ligand with high anity for the analyte. In Figure 5.10, examples are shown of dissociation in the presence of increasing amounts of competing ligand for a monovalent Lck protein and bivalent binding Syk protein.
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Figure 5.10
Eect of dierent concentrations of competing peptide ligand on the dissociation rate. (A) 200 nmol l1 Lck SH2 GST fusion protein with EPQpYEEIPIYL peptide and (B) 5 nmol l1 Syk tandem SH2 domain with g-ITAM peptide. Reprinted from ref. [11], Copyright (2000), with permission from Elsevier (A) and from ref. [14], with permission from Wiley-VCH (B).
The eect of the ligands is dramatic and illustrates that for a transport-limited interaction, the o-rate can be several orders larger than expected from the dissociation phase without competing ligand. Although the anity of these proteins for the ligands is rather high, relatively high concentrations are needed to prevent rebinding completely. In control experiments with high concentrations (4200 mmol l1) of non-binding peptides, the dissociation rate is at maximum, only a bulk eect, a higher baseline is seen, as also occurs in Figure 5.10B, for 4.4 104 mol l1 ITAM peptide. At high concentration of binding peptide the dissociation phase approaches a monophasic exponential decay (see Figure 5.11) and the curve can be tted with eq. (5.6) to derive the o-rate. In both cases the dissociation rate is very high. For the Lck protein, rebinding (Figure 5.11A) still seems not to be completely suppressed in the presence of even 104 mol l1 peptide. Especially at low R values with more free sites on the surface available for rebinding [see model eq. (5.8)], deviation from rst-order decay kinetics is observed; however, the rst ca. 80% of the decay can be approximated by the exponential function. The half-lifetime is about 1 s and ko is 0.6 0.1 s1, close to the value obtained from the rebinding model with xed experimental value for kon/ktr (0.38 s1; see Section 5.3.2.1). The dissociation of the Syk protein (Figure 5.11B) is also speeded up in the presence of competing peptide and shows monophasic exponential decay, with ko 0.13 0.02 s1, close to that of comparable mono- and bivalent interactions involving SH2 domains [25,26]. The high dissociation rate in combination with the high anity (KD 5 nmol l1) is intriguing. As a rule, high-anity monovalent interactions have slow dissociation rates, hence the complex has long half-lifetimes, e.g. for the avidinbiotin complex it is over 1 week [27]. The binding of a double phosphorylated ITAM-peptide with the tandem SH2
141
Figure 5.11
Dissociation of bound proteins in the presence of competing ligands to prevent rebinding. (A) Lck SH2 GST fusion protein in the presence of 100 mmol l1 peptide. (B) Syk tandem SH2 protein with 220 mmol l1 of bivalent binding ITAM-peptide. The monophasic-exponential or rstorder decay ts [eq. (5.6)] are indicated by the dotted lines. Reprinted from ref. [11], Copyright (2000), with permission from Elsevier (A).
domain is bivalent, existing of two weak monovalent interactions (see Section 5.4.1 for more structural details). For such multivalent interaction, the dissociation rate approaches that of a single (much weaker) monovalent interaction contributing to the bivalent binding, when competing (monovalent) ligand is present [28]. Therefore, in the presence of proper ligands, multivalent interactions oer the opportunity to combine high anity with high o-rates and short lifetime of the complex. In signal transduction processes, the combination of high anity and short half lifetimes might be decisive for specicity and transiency of proteinprotein interactions. It is remarkable that multivalent interactions, e.g. involving tandem-SH2 and tandem-SH3 domains, are abundant in signal transduction processes.
5.3.2.3
Experimental Procedure to Assay High O-rates
O-rates approaching 1 s1, as obtained in the previous section, are at the limit of what can be accurately measured by SPR. For really fast decay kinetics we think that the open cuvette structure is an advance as it allows direct accessibility for manual handling. Our protocol developed for assaying rapid dissociation kinetics in cuvette-based instruments starts with setting the instrumental sampling rate high (5 data points s1). The instrument is operated in one-channel mode. A 25 ml volume of the analyte protein, preferably with a concentration that saturates 490% of the binding sites,12 is pipetted manually into the sample cell. After reaching equilibrium of binding, the measurement is
12
This analyte concentration is approximately 10 KD.
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started and very quickly 10 ml of a high-concentration competing peptide is pipetted manually. The required concentration of peptide to prevent rebinding has to be determined experimentally (Figure 5.10). The data points of the resulting sensorgram can be exported, processed in a spreadsheet and tted to an exponential function. The data points within 1 s after peptide addition are discarded, as these are often aected by distortions. The experiment is repeated at least in triplicate with ample manual washing steps in between.
5.3.3 Quantitative Considerations on Mass Transport Limitation

As explained previously, MTL occurs if the reaction (binding) ux is much higher than the transport ux of analyte from the bulk solution to the sensor. These uxes are described by the transport coecient Lm and the Onsager coecient Lr for the reaction ux [21]. A quantitative measure for MTL is expressed in eq. (5.9). MTL Lr Lm Lr 5:9
If the analyte transport is totally rate limiting in the binding kinetics (Lr c Lm), MTL will approach 1. Lm is directly related to ktr as dened in model 2 (Scheme 5.1) for the association phase and eq. (5.8) for the dissociation phase. Lm in m s1 is obtained by conversion of ktr as indicated in Box 5.1 in Section 5.2.2.2. The Onsager coecient of reaction ux (Lr) in m s1 is obtained from eq. (5.10) [21]. Lr kon B
13
Here kon is converted to m mol s units. At the start of the interaction [B] equals Bmax, the maximum binding capacity of the sensor surface, i.e. the concentration of free analyte binding sites on the sensor surface. Bmax can be obtained from the Langmuir binding isotherm [eq. (5.2)] or from global kinetic analysis in m1. Using the same approach as explained in Box 5.1 Bmax is converted to [B] in mol m2 with eq. (5.11). B Bmax 103 mol m2 122 MW 5:11
1 1
5:10
The extent of MTL allows one to consider whether MTL can be avoided by adaptation of the experimental conditions, e.g. by lowering the binding capacity on the sensor surface or increasing the diusion rate by increasing ow or agitation of the bulk solution in the cell. The global kinetic analysis with model 2 yields Lr 1.72 103 m s1 and Lm 6.3 106 m s1 for the interaction with v-Src SH2 as analyte14
13 14
103 times kon in l mol1 s1. MW 12.3 kDa.
143
(see Figure 5.5). Applying eq. (5.9), under these conditions, MTL is practically 1 for this rather high binding capacity surface (Bmax 320 m1). To reduce MTL to 0.5 (Lr Lm), the binding capacity Bmax should be reduced to about 1 m1, which is not a feasible assay condition. Increasing ow and agitation will not be sucient, either. Assuming that a 5-fold increase in Lm can be obtained, the interaction will still be completely transport limited. In practice, the increase in Lm will be rather limited due to hydrodynamics (stagnant layer) and the dimensions of the (ow) cells and because diusion within the dextran matrix on the sensor is not sensitive for the ow conditions [23]. In general, for an analyte protein of approximately 40 kDa molecular weight, Lm is around 3 106 m s1. This number can vary somewhat depending on type of sensor chip. Applying eq. (5.9), this implies that for a surface with Bmax 100 m1 (corresponding to 2 108 mol m2) that Lm 4 Lr if kono2 102 m3 mol1 s1, corresponding to kono2 105 l mol1 s1. This number agrees very well with predictions from various MTL models [17,29]. The size of the analyte, of course, inuences the diusion rate and the kon value, but as a rule of thumb, transport limitation will aect binding kinetics to a dextran-based sensor surface, if kon is larger than 105 l mol1 s1. In summary, lowering the binding capacity and increasing the ow rate can prevent MTL only in the case of slightly transport-inuenced kinetics. In practice, we assume that the Lm/Lr ratio can be improved at most by about a factor 5 on changing the experimental conditions. As a consequence, in moderately and severely transport-limited cases an eect of MTL on the kinetics cannot be avoided.
5.3.3.1
Flow or Cuvette?
One can ask whether a ow or cuvette instrument is to be preferred when it comes to transport limitation. Although dierences in hydrodynamic behavior may exist between a ow and a cuvette instrument and detailed hydrodynamic models have been derived for ow cells [23,24], in practice, no signicant dierences in transport uxes between ow and cuvette systems have been observed. This is illustrated by the agreement of the Lm value of 9.8 106 m s1 for IL-2 (MW 14 kDa) obtained in a ow-instrument (Biacore) using a model similar to model 2 [30] and the value of Lm 6.5 106 m s1 for the v-Src SH2 domain (MW 12.3 kDa) obtained in a cuvette instrument (Table 5.1). We conclude that in practice no signicant dierences exist in the extent of MTL between cuvette and ow instruments.
5.4 Global Kinetic Analysis of Complex Binding Models

After describing simple bimolecular interactions, we shift to more complex binding mechanisms with conformational changes, dimerization, multicomponent interactions, multivalent binding, etc. In addition to structural
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information as derived from NMR and X-ray analysis, kinetic information and insight into the mechanism is valuable for understanding the binding process and is of special interest for rational drug design. We describe examples of applications of global kinetic analysis with more complex models to illustrate this point.
5.4.1 Global Kinetic Analysis Including Mass Transport and a Conformational Change
For better understanding of our rst example, the bivalent binding of Syk tandem SH2 domain (Syk tSH2) with a surface loaded with ITAM-peptide, we start with the description of the structural aspects. The interaction of an ITAMderived ligand with Syk tSH2 involves bivalent binding of two phosphotyrosine containing sequences on the ITAM-peptide with the two SH2 domains of the Syk protein. This interaction plays an important role in, amongst others, signal transduction of the IgE receptor (FceRI) and the B-cell antigen receptor [31]. An X-ray structure of Syk tSH2 with an ITAM peptide is shown in Figure 5.12. The linker part in the ITAM, between the two phosphotyrosine-containing sequences, hardly interacts with the protein [32]. The two SH2 domains in the
Figure 5.12
X-ray structure of Syk tandem SH2 domain (ribbon) with doubly tyrosine phosphorylated ITAM (sticks). Both phosphotyrosines are indicated in red. PDB entry 1A81 [32]. Reprinted with adaptation from ref. [14], with permission from Wiley-VCH.
145
Figure 5.13
Association phase sensorgrams for the interaction of Syk tandem SH2 domain with an immobilized ITAM peptide. Global kinetic analysis of the experimental data using CLAMP, according to the indicated models in red. Reprinted from ref. [14], with permission from Wiley-VCH.
Model 3: transport conformation model A0

k k
Model 4: transport dimer model

A0
k k
Model 5: 1PP-bivalency model

A+ B
k k k k
A
k k
A
k k
AB AB2
A+ B AB
k k
AB AB*
A+ B
AB A2 B
AB + B
AB + A
Scheme 5.2
Complex binding models used in global kinetic analysis of association phases.
Syk protein (labeled N-SH2 and C-SH2) are connected by a exible coiled coil linker, giving some exibility in the inter-SH2 distance. The association phase of this interaction was subjected to a global kinetic analysis (Figure 5.13). The association phase was assayed at dierent temperatures as part of a complete thermodynamic analysis (see Section 5.6 for thermodynamic analysis based on SPR). At 11 1C, deviation is observed using model 2 (Scheme 5.1) in global analysis in the association phase as when the signal approaches equilibrium. It is conceivable that a bivalent interaction occurs in two discrete steps [29], as indicated in Box 5.2. After initial monovalent binding, the second step involves a conformational (intramolecular) change, leading to a high-anity bivalently bound complex AB* (model 3, Scheme 5.2). This interaction is certainly transport limited, as we see a strong eect of added ligand on rebinding in the dissociation phase (Figure 5.10B). Also indicative of transport limitation is the initially linear association phase, especially at higher analyte concentrations [21]. Therefore, a transport step is included in model 3.
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Box 5.2 Relation of Kb, Kconf and Kobs in the conformation change model
Conformation change model 3 consists of two binding steps (see also Scheme 5.2): an initial binding event occurs, characterized by the equilibrium association constant Kb. Second, a structural change in the bound state occurs, characterized by equilibrium constant Kconf, leading to a higher anity complex. For a bivalent interaction as in the case of Syk t SH2 binding to doubly phosphorylated ITAM peptides, this last step includes a structural arrangement of the complex with a second intramolecular binding event.
Kb and Kconf are dened by eqs. (5.12) and (5.13): Kb AB1 AB 5:12
Kconf
kconf AB2 kconf AB1 AB1 AB2 AB
5:13
The observed equilibrium association constant Kobs is dened by eq. (5.14): Kobs 5:14
Note that [AB1] + [AB2] corresponds to the total amount of bound analyte, which is proportional to the change in SPR signal R. Substitution of eqs. (5.12) and (5.13) in eq. (5.14) yields Kobs Kb 1 Kconf 5:15
A plot of Req vs. [A] will also for this case obey a binding isotherm t as demonstrated in Figure 5.3, and from the t Bmax and Kobs are obtained.
Applying model 3 gives an excellent t (Figure 5.13B); in the ts, Bmax and ko were kept at experimental values.15 The t yields the parameters ktr, kon, kconf and kconf; the values especially of kon, kconf and kconf may not be reliable as they are strongly correlated (see Table 5.2). According to model 3, Kobs contains contributions of the initial binding step characterized by association constant Kb
15
Bmax is derived from the Langmuir binding isotherm [eq. (5.2)], which also holds for model 3 (see Box 5.2); ko is derived from the experiment shown in Figure 5.11B.
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Table 5.2
Parameters and their correlation from calculations used in Figure 5.13B.

Value
1
Parameter 1 2 3 4 ktr (m1 s l mol ) kon (l mol1 s1) kconf (s1) kconf (s1)
1
Correlation 1
7
Correlation 2 1.000 1.000
Correlation 3 1.000
5.4 10 1.19 106 2.81 0.16
0.74 0.72 0.72
and of the second intramolecular binding step or conformation change Kconf (see Box 5.2). Notwithstanding uncertainty in the rate constants, a Monte Carlo run16 with CLAMP gives consistent values for Kb ( kon/ko) and Kconf ( kconf/ kconf) of 1.9 107 l mol1 and 18.5, respectively. This value of Kb is signicantly higher than the 7.7 105 l mol1 found for the monovalent binding of monophosphorylated ITAM peptide [33]. It is likely that the two binding steps in model 3 are not completely resolved. First, no obvious biphasic association phase exists in Figure 5.13; second, by increasing the temperature above 30 1C, we obtain an excellent t using the bimolecular transport model 2 (Figure 5.13C), indicating that the two binding steps can no longer be discerned. Calculation of Kobs from the t parameters using eq. (5.15) (Box 5.2) yields a value of 3.6 108 l mol1, in excellent agreement with Kobs obtained from the binding isotherm at 11 1C (3.4 108 l mol1). Although the values obtained for Kb and Kconf may not be physically meaningful, they can be used to calculate solid Kobs values with eq. (5.15).
5.4.2 Unusual Kinetics: Intermolecular Bivalent Binding to the Sensor Surface

A second example from our work where global kinetic analysis plays a central role in elucidating the binding mechanism to a SPR sensor surface is Grb2 protein binding to an immobilized bivalent polyproline (PP) peptide (2PP). 2PP contains two PP binding epitopes derived from the SOS protein17 separated by a short linker moiety. The Grb2 protein exists of two SH3 domains and one SH2 domain connected by two exible linkers [34]. The two SH3 domains can each bind to one of the PP epitopes of 2PP in a bivalent mode [34,35]. In Figure 5.14, a model of the bivalent complex of Grb2 protein with 2PP is shown. It was expected that the kinetics of this bivalent interaction could be described by a model similar to that used for the Syk tSH2-ITAM interaction (Section 5.4.1); instead, a dierent binding model emerged. The binding of Grb2 to immobilized 2PP SPR sensor surfaces with dierent binding capacities is shown in Figure 5.15. The form of the curve describing the association phase
16
In a Monte Carlo run the t is repeated for a dened number of cycles with variation of the start parameters within a dened range. 17 The SOS-Grb2 interaction plays an important role in the signal transduction cascade of numerous receptors, controlling among others cell proliferation and dierentiation, platelet aggregation and T-cell activation [36].
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Figure 5.14
Model based on X-ray structure of Grb2 (PDB entry: 1GRI, ribbons) which a double polyproline peptide docked on the SH3 domains (sticks).
Figure 5.15
Sensorgrams of Grb2 protein (range 3302000 nmol l1) binding to immobilized 2PP-peptide with various binding capacities (Table 5.3). (A) and (B) net signal (reference cell subtracted from sample cell); (C) data from the sample cell only due to non-specic binding in the reference cell; lowest curve is the baseline not containing Grb2 protein.
appears to be sensitive to the binding capacity: at high binding capacities, the association phase looks conventional with a steady increase until equilibrium is reached. Lowering the binding capacity yields biphasic association, with a very fast initial increase, taking only a few seconds, followed by a slower increase (Figure 5.15B and C). The SPR signals at equilibrium apparently comply with the Langmuir binding isotherm [eq. (5.2)], as shown in Figure 5.16 for medium binding capacity. At high and medium capacity surfaces the binding isotherm
149
Figure 5.16
Equilibrium analysis with binding isotherm of Grb2 protein to a medium high capacity surface of 2PP peptide (data from Figure 5.15B).
Table 5.3
Data from binding isotherm of Grb2 binding to 2PP surfaces.

Bmax (m1) 1520 55 218 3 73 3 KD (nmol l1) 535 40 460 30 NDa
Binding capacity High Medium Low

a
ND Not Determined (equilibrium not reached).
gave comparable KD values (Table 5.3). At low binding capacities equilibrium is not reached within 20 min (Figure 5.15C). The data from binding of Grb2 to the 2PP surfaces with various binding capacities have been subjected to global kinetic analysis, exploring several models. As shown in Figure 5.17A, the data from very high binding capacity (Figure 5.15A) could be readily tted with a conformation change model (model 3, Scheme 5.2). Interestingly, the transport step could be omitted from the model, giving identical results. This suggests that in this case the interaction is not transport limited, in agreement with the calculated value of kon 5.8 104 l mol1 s1, which is below the indicated value for transport limitation (Section 5.3.3). At medium high and low capacity surfaces, model 3 shows systematic deviations from the experimental data as shown in Figure 5.17B: the slope of the slow phase in the ts changes much less with the concentration than experimentally observed. X-ray structures suggest that Grb2 dimers can be formed [37,38] and therefore the data were tted with dimer model 4 (Scheme 5.2), as can be seen in
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Figure 5.17
Global kinetic analysis of Grb2 binding to 2PP surfaces with CLAMP. (A) High binding capacity surface, tted with conformation change model 3. (B) Medium capacity surface, tted with conformation change model 3. (C) Medium capacity surface, tted with dimer model 4. (D) Low binding capacity surface, tted with dimer model 4. Details of these models are given in Scheme 5.2.
Figure 5.17C. The residual sum of squares parameter from the t with model 4 was 1.71, compared with 2.50 for model 3. Also for the low capacity surface tting with model 4 gave good results (Figure 5.17D). According to model 4 binding of the rst Grb2 molecule (A in the model) to immobilized 2PP (B in the model) facilitates binding of a second Grb2 molecule. However, we doubt that on the surface such physical Grb2 dimer will be formed, because we cannot demonstrate the formation of dimers in solution upon addition of 2PP-peptide to Grb2, either by chemical cross-linking or dynamic light scattering, in line with published results [37]. An alternative to bivalent intramolecular binding is intermolecular bivalent binding (see Scheme 5.3b). In the exible dextran matrix of the sensor chip, the distance between 2PP epitopes could easily adapt to facilitate intermolecular
151
Scheme 5.3
Schematic representation of various binding modes of Grb2 protein to 1PP and 2PP surfaces.
binding. If intermolecular bivalent binding occurs, this should be observed for a monovalent 1PP surface (Scheme 5.3c). In Figure 5.18, the association kinetics of 1PP surfaces with low and high binding capacity are shown. The curves can be readily approximated with a bivalent binding model: rst monovalent binding of Grb2 protein to immobilized 1PP, followed by bivalent binding to a second free 1PP ligand (model 5, Scheme 5.2). As expected, the rate of the slow bivalent binding step depends on the binding capacity: with a high capacity it will be easier to nd a second 1PP ligand
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Figure 5.18
Association phase sensorgrams of Grb2 protein binding to a low (A) and high (B) capacity monovalent 1PP surface. Global kinetic analysis with CLAMP according to bivalent binding model 5 (see Scheme 5.2).
and the rate will be higher. Interestingly, the anity of the initial fast binding step as derived from the kinetic analysis is approximately 12 mmol l1, which is similar to the anity of monovalent binding of 1PP to Grb2 in solution [35]. How can the intermolecular bivalent binding mode to the 2PP surface be reconciled with the observed association kinetics which can be well described by the dimer model 4 (Figure 5.17C and D)? We propose a three-step mechanism as outlined in Scheme 5.4: the rst step is monovalent (or bivalent) binding of Grb2 to one 2PP ligand; after this, bivalent binding with another 2PP ligand occurs (model 5). This intramolecular binding prepares a perfect second docking site for another Grb2 molecule as the inter-PP distance is already optimal for bivalent binding. This model also explains why the slow phase is much more delayed in case of low binding capacity (Figure 5.17): it is more dicult to nd a second partner for divalent binding. The proposed binding model for the 2PP surface cannot be dened in all detail in the CLAMP program, e.g. no bivalent ligands can be dened and no discrimination between single and double occupation of two 2PP ligands can be made. Actually, model 4 is a simplied approximation of the possible binding modes. It is possible that in the dimer complex (step 3, Scheme 5.4) dimeric interactions, as found in the X-ray structure of Grb2 are involved, as the Grb2 molecules are forced to be together. For the monovalent 1PP surface, such dimer formation cannot take place and the kinetics can be adequately described with model 5.
5.4.3 Global Kinetic Analysis: Concluding Remarks

At rst sight, in both examples in this section we have a protein that binds bivalently to an immobilized ligand. However, the outcome is surprisingly dierent. This is illustrative for the use of models in global kinetic analysis: one
153
Scheme 5.4
Sequential steps in the proposed model for binding of Grb2 to the 2PP surface, ultimately leading to the formation of dimers.
should be very careful in the interpretation of the applied binding model. In complex situations, good ts of the experimental curves can be derived from more than one model. This raises the question of how far the physical reality is reected by these models. A model will only provide an approximation: not all reactions can be included in detail, as described above. Unless the kinetic steps are well resolved, the calculated kinetic parameters may not be reliable, as they will also be strongly correlated. Hence it is useful to introduce all possible experimental values in the calculations. Other complications may arise from the fact that not all ligands are equally accessible: the sensor with immobilized ligand comprises a three-dimensional
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volume. Especially for high binding capacity sensors, partition into this volume may be hampered near saturation of binding due to crowding [23]. With multivalent binding analytes the hydrogel of the dextran on the sensor may become more cross-linked, leading to a more compact structure during the binding process. As the SPR signal decreases exponentially with distance from the gold surface (Chapter 2), this might lead to an accounted signal increase vs. the model. In spite of all these considerations, sometimes the quality of the ts with complicated binding models can be stunning (Figures 5.17 and 5.18). As a rule, a proposed binding model should be simple and supported by experimental evidence; additionally, it should include all possible xed experimental parameters. Global kinetic analysis is a unique tool, providing insight into the binding mechanism, the kinetics of an interaction and the role of protein dynamics. It can inspire new ideas for molecular design and drug development, for example, the length and rigidity of the linker between the two phosphotyrosinecontaining binding epitopes in ITAM-mimetic constructs binding to Syk tSH2 [14]. Ample examples exist using the simple bimolecular models 1 or 2; applications of more complicated models are rather scarce. Such examples are the binding of IL-2 to the heterodimeric IL-2 receptor [30], binding to a heterogeneous surface with two dierent ligands [39] and the kinetic analysis of amyloid bril elongation [40]. Deviation from the simple 1:1 model is already indicative of a more complex binding mechanism.
5.5 Anity in Solution Versus Anity at the Surface

In SPR measurements, interactions take place at the sensor surface, which is not always representative of interactions in solution. This is certainly true for divalent analytes, such as antibodies and GST fusion proteins that form dimers and show an avidity eect when binding to a surface [41]. The amount of analyte binding to the sensor surface in the presence of a competing ligand in solution is inuenced by the anity of the analyte for this ligand. If the anity is high, a relatively large amount of analyte will be in complex with the ligand in solution and only a small amount of analyte will be available for binding to the surface, resulting in a lower shift in SPR angle. Using this model, Morelock et al. developed a method to obtain thermodynamic binding constants in solution [42]. Based on this, we derived a tting model for data from competition experiments with constant analyte and varying ligand concentrations in solution (Box 5.3) [43]. An example of competition experiments is shown in Figure 5.19. Experiments were performed at various pH values to determine the shift in pKa of the phosphotyrosine upon binding [43]. The equilibrium dissociation constant at the chip (KC) was determined at each pH and these values were used in the ts. The experimental data was tted with eq. (5.19) (Box 5.3), using experimental values for [A]tot and KC, while the independent variable in the t is the ligand concentration [B]tot.
155
Box 5.3 Thermodynamic binding constants for binding in solution

In an SPR competition experiment with ligands for the analyte present both on the sensor surface and in solution, the two binding equilibria are as follows: 1. Interaction between analyte A and immobilized ligand B on the sensor chip (Bc), yielding complex ABc on the sensor. The dissociation constant (KC) is KC ABc ABc 5:16
[Bc] and [ABc] are in millidegrees; when all Bc sites are occupied [ABc] Bmax. 2. Interaction in solution between ligand B in solution with A to form complex AB. The dissociation constant (KS) is AB 5:17 AB Note that KS is a thermodynamic binding constant. In analogy with eq. (5.2), the amount of binding onto the surface can be described by a binding isotherm: Z Bmax 5:18 Req KC Z KS where [Z] is the total concentration of analyte [A]tot minus the amount of analyte in the complex AB ([Z] [A]tot [AB]), and eq. (5.18) changes to ! Atot AB Bmax 5:19 Req KC Atot AB The amount of complex AB in solution is a function of the anity in solution (KS) and eq. (5.17) can be rewritten: Atot AB Btot AB KS 5:20 AB
From this equation, it appears that [AB] is a quadratic function of the type ax2 + bx + c 0, for which the solution is given by the square root equation q 2 KS Atot Btot KS Atot Btot 4Atot Btot 5:21 AB 2 Substituting eq. (5.21) for [AB] into eq. (5.19) yields an equation that ts data from competition experiments. Fitting with [A]tot kept at the experimental value and [B]tot as independent variable provides KS and Bmax. Note that eq. (5.19) contains KC. The value of KC is obtained from a separate experiment. Best t is expected when [A]tot Z KC. Bmax is the maximum binding capacity upon complete saturation, and not the binding capacity in the absence of competing ligand.
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Figure 5.19
Data from SPR competition experiments to determine the binding constant KS in solution. The analyte is Lck-SH2 GST fusion protein (50 nmol l1), the immobilized ligand and the ligand in solution are identical [a phosphotyrosine 11-mer peptide derived from the hamster middle-T-antigen (hmT)]. Experiments were at dierent pH: from left to right pH 9, 6.8 and 5. The lines are the ts with the substituted eq. (5.19) (see Box 5.3). Reprinted from ref. [43], Copyright (2002), with permission from Elsevier.
In order to verify the reliability of our approach for obtaining anity data in solution and to see if anity at the sensor surface is signicantly dierent from that in solution, KC and KS values are compared in Table 5.4. The data illustrate that the anity of dimer proteins (the GST fusion protein and not-heated Grb2-SH2; see below) at the surface is larger than in solution. This can be explained by the avidity eect, occurring when the dimer binds bivalently to two ligands at the surface. The case of the Grb2-SH2 protein without GST part is interesting. It has been reported that this protein occurs as a dimer.18 Probably this is an artifact due to the expression as a GST fusion protein, which is known to form dimers through the GST part [44]. From size-exclusion chromatography we estimate that our GST-cleaved Grb2-SH2 protein contains B60% dimer. The dimer is metastable and upon heating to 50 1C the monomer is irreversibly formed [38]. Before heating, the anity to the sensor surface is higher, due to the large amount of dimer. KS is higher before heating, suggesting that the anity of the ligand for the dimer in solution is lower than for the monomer. For the pYVNV-peptide binding to monomer Grb2-SH2, consistent values for KS are obtained (230260 nmol l1), notwithstanding large dierences in KC (7.9 for the GST fusion protein to 790 for full-length Grb2) used in the
18
Dimer formation by domain swapping of an a-helix [38].
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Table 5.4
Comparison of anity at the sensor surface (KC) and in solution (KS), calculated from competition experiments using the substituted eq. (5.19) (see Box 5.3).
KC (nmol l1) 6 7.9 134 220 790 5.4 220 KS (nmol l1) 60 260 1800 255 230 5.7 234
Protein+peptide Lck-SH2 GST fusion proteina + hmT-peptide Grb2-SH2 GST fusion protein + pYVNV-peptide Grb2-SH2 not heatedb + pYVNV-peptide Grb2-SH2 heated + pYVNV-peptide Full length Grb2 protein + pYVNV-peptide Syk tSH2 + g-ITAM-peptide v-Src SH2 + hmT-peptide
a b
If explicitly indicated the dimer forming GST moiety is present. Heating to 50 1C converts the Grb2-SH2 dimer irreversibly to the monomer (see text).
calculations to obtain KS. Moreover, the solution anities agree with literature values obtained using other techniques. This strengthens our condence in the competition approach. As a rule, the anity of monomer proteins in solution is the same as at the sensor surface, even for the bivalent binding Syk tSH2! An exception seems to be the binding of the full-length Grb2 protein to the SH2 domain.19 In the case of a bivalent binding analyte with two (identical) binding sites such as an antibody, the expression for [AB] will be dierent from eq. (5.21). Now we have to take into account that not all occupied antibody remains in solution, as monovalently occupied antibodies are able to bind to the sensor surface. When a certain fraction of all binding sites are occupied, a statistically determined distribution exists over double-bound, single-bound and unbound antibodies in solution. Unbound antibodies, and also a single-bound antibody with a ligand from solution, can bind to the sensor surface. We have adapted the expression for [AB] (Box 5.3) to the statistical distribution [45] and it appears that this correction has only a modest eect on the resulting KS value (o10%). In summary, the approach derived in Box 5.3 cannot be used for every binding model. Especially when the Langmuir binding isotherm is not suitable for tting Req as a function of analyte concentration, this approach will not be valid. However, for more complicated binding models obeying the Langmuir binding isotherm, such as the two-step model proposed for Syk tSH2 (Box 5.2), reliable KS values can be obtained. In this case KS will be an apparent binding constant, containing the various contributions to Kobs (see Box 5.2). The competition experiments as described in this section are very attractive in drug research: the anity of a range of potential drug candidates can be assayed at the same surface! In general the standard error in KS is larger than in KC. Processes in solution may not always be representative for processes at sensor surfaces or in biological systems. We are convinced that in some cases interaction at a surface might be a better model than interaction in solution,
19
A possible explanation for this dierence is discussed in ref. [12].
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especially with multivalent interactions. For example, the Sos-protein20 contains multiple (six) polyproline sequences to recognize Grb2 SH3 domains [36]. Several Grb2 molecules might bind bivalently to these sequences in one Sos molecule in dierent combinations. For this a surface loaded with polyproline ligands might be a better model than 1:1 interactions in solution.
5.6 Thermodynamic vant Ho Analysis Using SPR Data

As described in the Introduction, it is no longer opportune to describe ligand receptor interactions in terms of a rigid lock-and-key concept. Binding of a receptor by a ligand can inuence the dynamics, induce allosteric changes of the receptor or, very importantly, have an eect on bound water molecules. All this can be vital for the biological eects in a biomolecular interaction. In this section we will concentrate on SPR-based assay of thermodynamic parameters, to reveal the biomolecular recognition process, to help understand it and to exploit it for improved rational drug design (see Box 5.4) [5,6,8].
5.6.1 vant Ho Thermodynamic Analysis

vant Ho thermodynamic analysis requires the measurement of the anity at a range of temperatures. As the SPR signal is extremely sensitive to temperature changes, complications may arise when measuring at temperatures deviating from room temperature, as some time may be needed before complete thermal equilibration is reached. An example is shown in Figure 5.20: in the reference cell a temperature eect is observed in addition to a bulk eect. It takes about 100 s to reach thermal equilibrium. The temperature eect is not visible in the net signal (Figure 5.20). The best approach is to use Req for the anity assay as by the time Req is reached the system will be in thermal equilibrium. It is important, especially when kinetics are assayed, that the sample is at the correct temperature and that the injection system is well thermostated. The design of newer generations of SPR instruments, e.g. Autolab ESPRIT, is optimized for anity assays in a temperature range 1045 1C. The simplest case of binding is a bimolecular interaction, such as that between v-Src SH2 and hmT-peptide as described above. The vant Ho thermodynamic analysis of this interaction is shown in Figure 5.21. The data can be readily tted with eq. (5.26) and the resulting thermodynamic parameters are included in Table 5.5. The data show that the anity at the sensor surface (KC) matches that in solution (KS), using the method described in Section 5.5. The convex form of the curve indicates a negative value for the heat capacity (DCp) and this interaction appears to be enthalpy driven. A concise description of how to interpret thermodynamic parameters in terms of molecular events during the binding process is given in Box 5.5. A more detailed interpretation can be found in ref. [12].
20
This is a crucial interaction in the activation of the Ras signaling pathway described in ref. [36].
159
Box 5.4 Thermodynamics of binding

For the simple bimolecular interaction between A and B yielding the complex AB, the change in Gibbs free energy (DG) is related to the standard Gibbs energy change under standard conditions (1 mol l1 of A and 1 mol l1 of B, at 25 1C): AB RT ln Ka RT ln Kd DG DG RT ln 5:22 AB where R is the gas constant and T is the absolute temperature. DG1 consists of a heat component released or taken up during the binding process (enthalpy, DH1), and an entropy (DS1) component related to the change in the degree of order of the system due to binding: DG DH T DS RT ln Ka 5:23
For proteinprotein interactions and other biomolecular interactions DH1 and DS1 change with temperature. The temperature dependence of DH1 and DS1 can be described in terms of the heat capacity (DCp) as given in eqs. (5.24) and (5.25): DH DH T DCp T T DS DS T DCp lnT =T 5:24 5:25
DCp is assumed constant within the applied temperature range. T1 is the reference temperature, usually 25 1C, and DH1(T1) and DS1(T1) are the values of DH1 and DS1 at this temperature. From eqs. (5.23)(5.25) follows the integrated vant Ho equation, eq. (5.26), which describes the temperature dependence of the anity constant KA: ! DH T DS T DCp T T T ln 5:26 ln KA RT R T R T This expression can be used to t KA values derived at various temperatures versus 1/T, and yields the thermodynamic parameters DH1, DS1 and DCp. A concise description of the interpretation of these parameters in terms of molecular events related to the binding process is given in Box 5.5.
Box 5.5 How to interpret thermodynamic binding parameters?

It is important to realize that in a thermodynamic analysis two situations are compared: the situation after the process (e.g. binding) is completed, vs. the situation before the process. This is why we look at the dierence in the thermodynamic parameters (indicated as D) including the whole of the process, e.g. also the solvent. The thermodynamic analysis of a single interaction usually tells us whether the binding is entropy or enthalpy driven, but it is not possible to interpret molecular processes more in detail, e.g. whether replacement of water molecules is involved or whether protein dynamics decreases. The power of thermodynamic analysis for drug design lies in the combination of 3D structural information and the study of
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structurally related compounds. This can give detailed insight on how specic structural features contribute to binding energetics. The most important thermodynamic parameters in molecular structural events are: DH binding enthalpy represents the heat eects involved in the interaction. It can be directly experimentally determined with calorimetric measurements. The heat eects are caused by the formation and disruption of non-covalent bonds (hydrogen and ionic bonds and van der Waals interactions) and can involve bonds between the reactants, but also bonds of solvent reorganization and conformational rearrangements of the reactants during the binding process. A large part of DH is due to bulk hydration. In drug design, more water molecules at the interaction interface may extend the complementarity of the surfaces and H-bond networks [9]. This is favorable for enthalpy, but disadvantageous due to a loss in entropy, and contributes to the phenomenon of entropyenthalpy compensation (see text). DS binding entropy can in general be interpreted in terms of degree of order and disorder of the system. This might comprise designed restriction of conformational freedom and rotation of chemical bonds involved in binding. Also, hydration can be a major factor for entropy, e.g. in hydrophobic binding: the burial of water-accessible surfaces and resulting release of water molecules can contribute to binding due to increases in entropy. DCp heat capacity is almost entirely ascribed to solvent eects and is considered of high information content. DCp can be determined directly in calorimetric experiments over a temperature range. DCp can be interpreted in terms of solvent-accessible hydrophobic and polar surface areas, buried in the binding process [5]. A decrease in accessible hydrophobic surface upon binding has a negative eect on DCp; that of polar surface a positive eect. Experimental DCp values have been related to dierent degrees of success to 3D structural information on these surface changes upon binding. Problems arise due to solvation eects and release or ordering of water molecules during binding. Ordering of water molecules in the binding interface has a large negative contribution to DCp [4].
Figure 5.20
Temperature eect on SPR sensorgrams. Left: signals of sample cell (solid line) and reference cell (broken line). Right: net signal (reference cell minus sample cell). Reprinted from ref. [46], Copyright (2003), with permission from Elsevier.
161
Figure 5.21
vant Ho plot for binding of hmT-peptide with v-Src SH2 domain. (J) Anity at the sensor surface, KC; (K) in solution, KS. The lines are calculated using eq. (5.26). Reprinted with permission from ref. [12], Copyright (2005) American Chemical Society.
Table 5.5
Parameter
Thermodynamic parameters derived from vant Ho thermodynamic analysis shown in Figure 5.21.
v-Src SH2 with 11-mer hmT-peptide
1 a
DH1 (kcal mol ) TDS1 (kcal mol1)a DCp (cal mol1 K1) DG1 (kcal mol1)a KD (nmol l1)
a
9.4 0.6 0.3 0.6 920 160 9.1 220 30
At reference temperature 25 1C.
5.6.2 Comparison of SPR Thermodynamics with Calorimetry

The heat eects of an interaction can be directly measured using calorimetry. Especially the introduction of isothermal titration microcalorimetry (ITC) instruments with improved sensitivity has greatly advanced the use of calorimetry in biomolecular interactions [9]. A debate is ongoing on the equivalence of enthalpy values from vant Ho analysis (DH1vH), compared to those from calorimetry (DH1cal): discrepancies between DH1vH, from several techniques for anity assay and DH1cal have frequently been observed [4750]. In calorimetric assays the total of the heat eects is assayed, e.g. heat of dilution, of mixing and heat eects due to changes in buer protonation and solvent equilibria linked with the binding process [47], which go beyond the intrinsic enthalpy contribution of the simple equilibrium A + B " AB. On the other hand, linked equilibria like that of buers and solvent will also inuence the anity and
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DH1vH. The situation may even become more complicated when DCp is not constant with temperature which can occur in multi-step binding processes. Horn et al. demonstrated that, when experimental setup and analysis are performed correctly, there is no statistically signicant dierence between DH1 values [51]. This holds even for complicated binding models including a conformational equilibrium as shown in Box 5.2. It should be remarked that vant Ho analysis is peculiar in its error estimation. Recently, Tellinghuisen demonstrated that the usual way of error estimation in vant Ho analysis is actually not correct and that the errors in DG1, DH1, DS1 and DCp are a function of temperature, leading to relatively large errors in DCp [52]. The number of thermodynamic studies using SPR is rather limited [12,14,50,5355]. In our experience, the thermodynamic parameters from SPR vant Ho analysis often compare fairly well with those from ITC [12,14]. As an example, in Figure 5.22 we show the match between our SPR data and ITC data from various studies in an entropyenthalpy compensation (EEC) plot for a wide range of ligands for the Lck and v-Src SH2 domains [46]. EEC is a universal phenomenon in biomolecular interactions in water and is generally a problem for the medicinal chemist as a gain in enthalpy, e.g. by adding hydrogen bonds to strengthen the binding, will be counteracted by a loss in entropy [56].
Figure 5.22
Entropyenthalpy plot for binding of various ligands to the Src- or Lck-SH2 domains. Open symbols: data derived from various ITC studies. Closed circles: data derived from SPR competition experiments, with peptide and peptoid ligands. Reprinted from ref. [46], Copyright (2003), with permission from Elsevier.
163
Combining SPR and calorimetry to explore fully the thermodynamics and kinetics of interacting systems might provide an optimal approach. To explore the information content of especially DCp values fully, ITC is generally a better choice than SPR thermodynamic analysis. A disadvantage of ITC is that it requires much more material (at least 100 times as much as is needed for SPR). The strong point of the SPR technique is the anity data and kinetics derived from the same data set.
5.6.3 Transition State Analysis Using Eyring Plots

SPR analysis has the unique feature that kinetic and anity information can be obtained from one experiment. This implies that thermodynamic experiments can be performed by analyzing the temperature dependence of kon and ko. Eyrings transition state theory provides the fundamental conceptual framework for understanding rates of chemical processes [57]. The transition state (AB#) is the high energy state along the pathway of reactants to product (for a binding process, the unbound species to the complex). A B AB# ! AB
k1 k1 k2
5:27
Based on eq. (5.27), the thermodynamic equilibrium constant for formation of the transition state is dened as K# [AB#]/[A][B]. Applying statistical mechanics, we obtain the Eyring equation state that holds for a rate constant k: k kB T # K h 5:28
where kB is Boltzmanns constant (1.381 1023 J K1) and h is Planks constant (6.626 1034 J s). K# is related to DH# and DS# (the activation enthalpy and entropy, respectively) in the same way as KA is related to DH1 and DS1 [eqs. (5.22), (5.23) and (5.26)]. This implies that for a linear Eyring plot [ln(kh/kBT) vs. 1/T] the data can be tted with eq. (5.29). kh DH # 1 DS# 5:29 In kB T R T R A non-linear Eyring plot can be tted with eq. (5.26); such ts yield the # activation parameters DH#, DC# p and DS . Transition state analysis using Eyring plots derived from SPR data have been published elsewhere [12,14,53,54]; an example is given in Figure 5.23. The ko values at various temperatures were determined in the presence of high concentrations of competing ligand to prevent rebinding (Section 5.3.2.2). The kon values were derived from kon ko/KD. It appears that the Eyring plot for ko is linear, indicating that DC# p is zero between the complex and the transition state (vice versa). The plot for kon shows a convex curvature, indicating that DC# p for formation of the transition state from the reactants is not zero. DC# p has the same absolute value as that derived for DCp from the
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Figure 5.23
Eyring plots for the interaction of v-Src SH2 domain with hmT 11-mer phosphopeptide. (a) kon, tted with eq. (5.26); (b) ko, tted with equation (5.29). Reprinted with permission from ref. [12], Copyright (2005) American Chemical Society.
vant Ho analysis of KA for the same interaction (Figure 5.21), because going from the complex to the transition state (ko) DC# p is zero. The Eyring plot for kon (Figure 5.23a) is interesting as it displays non-Arrhenius kinetics above 20 1C, i.e. at higher temperature kon decreases. Non-Arrhenius kinetics have been frequently found for protein folding. A general explanation for this phenomenon is that at higher temperatures a wide region of conformational space is visited and the probability of a exible ligand or part of a protein having the proper conformation for binding or folding, decreases [58]. Such a model makes sense for the binding of a pYEEI-peptide to an SH2 domain, as the high-anity binding can be regarded as a two-pronged plug into a twoholed socket in need of suitable positioning of the pY and I residue for binding [59]. If, for instance, binding starts with the pY moiety in its binding pocket, at higher temperatures it will be more dicult to have the I residue in the correct position to allow high-anity binding. A transition state analysis can give additional information, as is also illustrated by the comparison of binding phosphotyrosine ligands to the v-Src SH2 domain vs. Grb2 protein. The anity and DG1 values are comparable, even the activation energies DG# are nearly identical (Figure 5.24). However, transition state analysis reveals large dierences in DH# and DS#. Both DH# values are negative, indicating that upon formation of the transition state, heat is released. The high barrier of activation energy is caused by unfavorable activation entropy contribution. For binding to v-Src SH2 this contribution is about 4 kcal mol1 more unfavorable. This means that formation of the transition state of the Src SH2 domain from the reactants involves a higher degree of ordering than that of Grb2 SH2. Upon binding, the dynamics of Grb2 and v-Src SH2 domains is decreased to the same extent, leaving the large dierence in DS# unexplained [12]. The dierence in thermodynamic behavior can probably be
165
Figure 5.24
Energy transitions at 25 1C as a function of the binding coordinate for phosphorylated peptide binding to v-Src SH2 domain (solid lines) and to Grb2 SH2 domain (dotted lines). Reprinted with permission from ref. [12], Copyright (2005) American Chemical Society.
attributed to the role of water molecules, which form a hydrogen bonding network at the binding interface between ligand and v-Src SH2 protein [7] upon formation of the transition state, which has an entropy price. On the other hand, the water molecules in the network make a favorable enthalpy contribution to the transition state, explaining the favorable DH#. For binding to Grb2 SH2, such a role of water molecules is not inferred, only direct contact exists between the ligand and the protein. The above described transition state analysis has been criticized, because it is assumed that every activation leads to complex formation [60]. Alternatively, Arrhenius analysis is advocated [60]. In the interpretation of the data, 3D information from X-ray and NMR analysis is essential. However, the 3D structures alone cannot provide information on energetic contributions determining the binding process. Especially in cases where dynamics of ligand and receptor or solvent eects are involved, results of computational chemistry can be expected to be disappointing. The contribution of entropy to the free binding energy can be very large and may inuence the anity by several orders of magnitude. Thermodynamic and kinetic analysis can help to quantify the extent of these contributions and to generate ideas to exploit them in molecular design.
5.7 SPR Applications in Pharma Research: Concluding Remarks and Future Perspectives
In this chapter, we have emphasized the role of kinetics and thermodynamics in biomolecular interactions. Notwithstanding the impressive contributions of structural biology and computational chemistry, our understanding and the ability to predict anities of receptorligand interactions remain poor. It is
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increasingly acknowledged that for a more accurate notion, thermodynamic and kinetic studies of biomolecular interactions are needed. Modern calorimetric and SPR techniques are the tools to perform such studies and deserve a place in the toolbox of rational design used by the medicinal chemist and chemical biologist. The high information content of SPR data with kinetic and anity information is unique and allows full thermodynamic and kinetic characterization of an interaction, including transition state analysis, as shown in Section 5.6.3. There are also limitations to the use of vant Ho analysis: even with anity data with relatively small standard errors, DCp has a large error. DCp is important as it can give insight into the nature of the surface area buried upon binding and on the role of water molecules in the binding process (Box 5.5). Using ITC, in general DCp can be more accurately determined by measuring DH at a wide range of temperatures. The best of both worlds is to use SPR and ITC for thermodynamic analysis, with special emphasis on careful experimental design and on the limitations of each method. A concern associated with SPR data is that anity for a ligand immobilized on a sensor surface might not be identical with that for the ligand in solution. In this respect it is relevant to introduce linkers between the binding epitope and the dextran matrix of the sensor chip, as described in this chapter. In many cases no dierence appears, especially for monovalent binding. Using the approach for assays of KS with competition experiments as outlined in Box 5.3, we nd comparable anities at the surface and in solution (see Table 5.4 and Figure 5.21). On the other hand, binding to a surface might be a better model for a biological interaction involving multivalency than (monovalent) interactions in solution. Apart from drugreceptor studies, SPR is also useful for other aspects of drug research. Studies on binding to serum proteins are relevant for distribution properties of drugs [61]. Many important drug targets are membrane-bound proteins, e.g. G-protein coupled receptors (GPCRs). Technology to follow passive and active absorption to membrane interfaces using SPR is under development, as is drug binding to metabolizing enzymes [62]. SPR biosensor systems with supported monolayers and tethered bilayer membranes are under development, but not standard technology yet [63]. In an approach called ligand shing, crude tissue extracts and cell homogenates are screened for potential ligands or targets using SPR [62]. In such approaches, identication of bound species is crucial, with mass spectrometry (MS) as the ideal platform. In particular, matrixassisted laser desorption/ionization time-of-ight MS (MALDI-TOFMS) and electrospray ionization MS (ESI-MS) are powerful tools for protein identication. It is therefore not surprising that it has been attempted to integrate SPR and MS for proteome analysis, as reviewed in ref. [64]. SPR serves two main purposes in proteome analysis: (1) to conrm and possibly quantify specic binding and (2) to act as a micro purication support for further analysis. MALDI analysis directly on a chip surface is possible [65]. Problems may arise due to the small amount of captured protein on the chip, the many handling steps of the procedure and the
167
acidity of the matrix material. In another approach, analytes are eluted and collected in a recovery system. In principle then the whole range of MS techniques, including analysis of digested samples, is available for identication. In drug research, high-throughput screening (HTS) plays an essential role in screening large libraries of compounds. Until recently, the use of SPR technology was hampered by the limited number of surfaces on one sensor chip. In view of the high commercial potential, in the slipstream of the development of array technologies, SPR imaging applications are emerging using microarrays on chips. Examples include Biacores Flexchip microarray device [66] and IBIS Technologies IBIS-iSPR instrument (see Chapter 6) and other SPR imaging techniques [67,68] (Genoptics and K-MAC, respectively). This eld is developing rapidly and is extremely promising. We expect an increasing impact of SPR technology on drug research. This will be enhanced by further developments of SPR technology for pharma applications, such as high-throughput screening, further integration of SPR and MS and mimicking membrane environments and protein ensembles on SPR surfaces.
5.8 Questions
1. What causes mass transport limitation (MTL) in the kinetics of SPR experiments? 2. How can MTL be diminished by experimental design? 3. How can one perform kinetic analysis under MTL conditions? 4. How can depletion of the analyte in a cuvette-based system be calculated? 5. What is the strength of SPR as a tool in drug development research? 6. Explain why a high loading of the ligand aects the determination of the anity constant. Describe at least two ways to solve this and determine from the sensorgram given below the anity constant of an antibody antigen reaction in nmol l1.
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5.9 Symbols
[A] [A]0 [A]free [A]tot [B] A AB# B Bmax D DCp DCp# DG DG# DG0 DH# DH0 DS# DS0 h K# KA kb KC kconf Kconf k-conf KD kobs ko kon KS ktr Lm Lr MW concentration of analyte A at the sensor surface (mol l1) initially added analyte concentration in the bulk (mol l1) analyte concentration corrected for depletion (mol l1) total analyte concentration used in a competition experiment (mol l1) concentration of free analyte binding sites on the sensor surface (mol m2 or m1) molecule A (usually analyte) transition state on formation of the AB complex molecule B (usually ligand) maximum binding capacity (in m1) diusion coecient (m2 s1) heat capacity (J mol1 K1) activation heat capacity (J mol1 K1) Gibbs free energy (of binding) under non-standard conditions (J mol1) Gibbs activation free energy for formation of the transition state (J mol1) Gibbs free energy (of binding) under standard conditions (J mol1) activation enthalpy (J mol1) change of enthalpy (upon binding) under standard conditions (J mol1) activation entropy (J mol1 K1) change of entropy (upon binding) under standard conditions (J mol1 K1) Plancks constant (6.6262 1034 J s) equilibrium association constant for formation of the transition state (l mol1) equilibrium association constant (l mol1) Boltzmans constant (1.381 1023 J K1) equilibrium dissociation constant for anity at the chip (mol l1) rate constant for conformation change AB -AB* (s1) equilibrium constant for conformation change (kconf/k-conf) rate constant for conformation change AB* -AB (s1) equilibrium dissociation constant (mol l1) kon[A] + ko (s1) dissociation rate constant (s1) association rate constants for formation of the complex AB (l mol1 s1) equilibrium dissociation constant in solution (mol l1) transport rate for diusion from bulk to sensor surface (in m1 s1 l mol1) transport coecient from bulk solution to sensor surface (m s1) Onsager coecient for reaction ux (m s1) molecular weight of analyte (Da)
169
R Req Rt S T T0 Vbulk Z
gas constant (8.3143 J K mol ) net increase in SPR angle at binding equilibrium (reference cell subtracted from sample cell (m1) net increase in SPR angle at time t (reference cell subtracted from sample cell (m1) surface of the sensor in the cells (mm2) absolute temperature (K) reference temperature (298 K) volume of the bulk solution in the cells (l) viscosity (cP)
5.10 Acknowledgements
The examples presented in this chapter originate from work in the Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, The Netherlands. We wish to thank the Head of the Department, Professor Rob M.J. Liskamp, for making it possible to study exciting new compounds in our SPR assays. Many members of the Department have contributed: we especially thank Dr. Frank J. Dekker, Dr. Rob Ruijtenbeek and Dr. Ir. John A.W. Kruijtzer. We are grateful for the Rene Descartes, Paris) and cooperation with Dr Isabelle Broutin (Universite Professor Albert Heck (Biomolecular Mass Spectrometry, Utrecht University). We thank The Netherlands Organization for Scientic Research (NWO) for nancial support.
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CHAPTER 5

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

5.2.1 Anity Constants Derived from Equilibrium SPR Signals

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

Correction for Depletion of Free Analyte Concentration in the Cuvette

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

Or even lower, depending on the sensitivity of the instrument.

kobs Kinetic Analysis

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

Global Kinetic Analysis with a Simple Bimolecular Binding Model

2: Bimolecular model + transport step

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

dA ktr A0 ktr A kon AB koff AB dt

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

5.3 Detecting Mass Transport Limitation: A Practical Approach

5.3.1 Eect of Viscosity Change on the Association Phase

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

5.3.2 Transport Limitation in the Dissociation Phase

Rebinding Model for Transport-limited Dissociation

Kindly provided by Dr. Peter Schuck.

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

Competing Ligand to Prevent Rebinding During Dissociation

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

Experimental Procedure to Assay High O-rates

This analyte concentration is approximately 10 KD.

5.3.3 Quantitative Considerations on Mass Transport Limitation

103 times kon in l mol1 s1. MW 12.3 kDa.

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

5.4 Global Kinetic Analysis of Complex Binding Models

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

Model 3: transport conformation model A0

Model 4: transport dimer model

Model 5: 1PP-bivalency model

Complex binding models used in global kinetic analysis of association phases.

kconf AB2 kconf AB1 AB1 AB2 AB

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

Parameters and their correlation from calculations used in Figure 5.13B.

Correlation 2 1.000 1.000

5.4 10 1.19 106 2.81 0.16

0.74 0.72 0.72

5.4.2 Unusual Kinetics: Intermolecular Bivalent Binding to the Sensor Surface

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

Data from binding isotherm of Grb2 binding to 2PP surfaces.

Binding capacity High Medium Low

ND Not Determined (equilibrium not reached).

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

5.4.3 Global Kinetic Analysis: Concluding Remarks

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

5.5 Anity in Solution Versus Anity at the Surface

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

Box 5.3 Thermodynamic binding constants for binding in solution

Dimer formation by domain swapping of an a-helix [38].

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

A possible explanation for this dierence is discussed in ref. [12].

5.6 Thermodynamic vant Ho Analysis Using SPR Data

5.6.1 vant Ho Thermodynamic Analysis

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

Box 5.4 Thermodynamics of binding

Box 5.5 How to interpret thermodynamic binding parameters?

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions

9.4 0.6 0.3 0.6 920 160 9.1 220 30

At reference temperature 25 1C.

5.6.2 Comparison of SPR Thermodynamics with Calorimetry

Kinetic and Thermodynamic Analysis of LigandReceptor Interactions