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F
α (F) = αoe F∆x /kT
Laser
Photodetector
∆x = ?
Cantilever
F
Silicon nitride tip
Multi-modular
protein Piezoelectric
positioner
Figure 1
The unfolding of protein domains by an external force. (a) When axial stress is applied to a folded domain the protein will unravel. The inset
shows an equation describing this transition, where F is the applied force, Dx is the distance over which the unfolding event occurs, a0 is the
rate constant in the absence of an applied force, k is Boltzmann’s constant and T is the absolute temperature. Thus, the rate at which pro-
tein unfolding occurs increases exponentially with the applied force. This equation is similar to that describing the dissociation of non-cova-
lent bonds placed under an external force38,39. (b) The force–extension mode of the atomic force microscope (AFM). When pressed against a
layer of protein attached to a substrate, the silicon nitride tip can adsorb a single protein molecule. Extension of a molecule by retraction of
the piezoelectric positioner results in deflection of the AFM cantilever. This deflection changes the angle of reflection of a laser beam striking
the cantilever, which is measured as the change in output from a photodetector.
The force–extension relationship of ECM protein tenascin20,21, which con- will be discussed below, the early un-
polymers need not, however, be solely tains repeats of the FN-III domain folding peaks show clear deviations
entropic. AFM studies of certain gluco- (Fig. 2c). The unfolding of each of the from the entropic behaviour predicted
pyranose polysaccharides showed that, FN-III domains can be described accu- by the WLC model. This force–extension
while their elastic behavior was en- rately using the WLC model. The mean curve thus illustrates an important
tropic under low force, a transition that force at which the domains unfold is drawback in the use of native protein
occurs at higher force results in an in- 137 pN and the mean interval between fragments for the study of mechanical
crease in contour length16–18. This was peaks is 24.8 6 2.3 nm (Ref. 20). properties. When pulling a hetero-
shown to be due to the conversion of AFM measurements offer an opportu- geneous multi-domain protein, one usu-
individual glucopyranose rings from the nity to understand the characteristics ally cannot know which unfolding peak
‘chair’ conformation, which is energeti- that underlie the mechanical properties corresponds to which domain. The elas-
cally favoured at low force, to the longer of proteins. Elongation of the cytoskel- tic properties of specific domains are
‘boat’ conformation17. An even more etal protein spectrin (Fig. 3a), which con- therefore difficult, or impossible even,
dramatic deviation from entropic elas- tains repeated a-helical domains, results to identify. However, as discussed below,
ticity is seen in the extension of multi- in a markedly different force–extension a solution to this problem has been
domain proteins. The force-extension curve22. Unfolding occurs at much lower provided by molecular biology.
curves of these proteins show peaks forces (25–35 pN) and the interval be-
that correspond to the unfolding of sin- tween peaks (31.7 6 0.3 nm) reflects the Mechanical amplification by polyprotein
gle domains (Fig. 2b). As these proteins larger number of amino acids within engineering
are elongated, the restoring force in- each spectrin domain23. Atomic force Force–extension curves for small or
creases. At a certain force, however, one microscopy might thus allow for the single-fold proteins are difficult to in-
of the domains in the chain unfolds. Like distinction between mechanical topol- terpret because non-specific interac-
the freeing of a tangle in a rope, this un- ogies of different domain types. The ex- tions between the cantilever tip and the
ravelling suddenly adds to the effective tension of some proteins, however, adsorbed protein layer can obscure the
length of the protein and allows the leads to results that are more complex. interactions of interest at short exten-
force on the cantilever to fall to near Force–extension curves for a fragment sions, and their rupture might result in
zero. Further extension is resisted again of titin consisting of Ig domains 27–34 ‘peaks’ that resemble unfolding events.
by entropic forces until a second (Fig. 3b) revealed up to eight unfolding A regularly spaced saw-tooth pattern of
domain in the chain unravels. The peaks (six in this example) at forces of peaks, however, is a clear indication
force–extension curve therefore dis- 150–300 pN (Ref. 19). The height of the that a single, multi-domain protein is
plays a characteristic saw-tooth pattern peaks tends to increase with each un- being stretched. Recombinant proteins
with the number of peaks correspond- folding event, which suggests that do- consisting of multiple repeats of a
ing to the number of domains stretched mains have different mechanical stabil- specific domain were therefore con-
between the substrate and cantilever ities and that those that are less structed. The first domain chosen for
tip. This phenomenon was first demon- mechanically stable unfold before those study was Ig domain I27 of titin19 be-
strated with titin19 and later with the that are more stable. Furthermore, as cause it has a tertiary structure that is
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TIBS 24 – OCTOBER 1999 REVIEWS
well defined by NMR (Ref. 24), a known p
thermodynamic stability25, and an un- (a)
folding pathway that has been modeled 2
using steered molecular dynamics26 (a kT 1 x 1 x
F(x) = 1− − +
method for predicting how a protein F p 4 Lc 4 Lc
structure will respond to applied force).
Polyproteins consisting of either eight x
or 12 repeats of this domain were
cloned and expressed (Fig. 4a,b)27. Lc
Electron microscopic imaging of rotary F
shadowed I2712 (12 repeats of Ig domain
I27) demonstrated that these proteins
have a rod-like structure with a length of
~58 nm (Fig. 4c), which is close to that (b)
4
expected based on NMR measurements
of a single domain (4.4 nm) (Ref. 28). In
contrast to the staircase pattern of un- 3
folding peaks seen with titin (Fig. 3b),
the force peaks for I27 unfolding were
not ordered and were distributed 2 4
around a single mean value of ~200 pN
(Fig. 4d). The fitting of consecutive 1
unfolding peaks according to the WLC
2
model showed that each unfolding
1
event added 28.4 6 0.3 nm to the length 3
200 pN
of the protein. Protein engineering has
therefore enabled precise measure-
ments of the length increment caused
by domain unfolding and of the mecha- 20 nm
nical stability of individual protein
domains. (c)
Lc (nm) 52.8 80.3 108.1 136.2 164.4 192.4 220.7 248.7
Kinetics of force-induced unfolding and p = 0.56 nm
refolding
The probability that a domain will un-
fold is dependent on the applied force,
the extension required to break the
100 pN
sequence between the A9 and G strand the I27 domain by the predicted 15% B
hydrogen bonds (72–78, depending on (Ref. 32). This deviation is particularly
how it is counted) agrees well with the evident during the domains that unfold Trends in Biochemical Sciences
observed space between peaks (28.4 6 first and is likely to represent simul-
0.3 nm), which predicts 75 amino acids taneous breakage of the A–B bonds in Figure 5
in the I27 fold (75 3 0.38 nm 5 28.5 nm). each of the domains. The appearance of The mechanical topology of I27. A
When mutant polyproteins were con- a smaller hump during the subsequent schematic diagram of the topology of the
structed with an extra five glycine unfolding event suggests that the re- b-sandwich structure of I27. Each b-
strand is shown as an arrow, with
residues within the fold, the interval be- maining domains rapidly return to their strands from the two b-sheets shown in
tween unfolding peaks was lengthened original conformation before the next different colours. The sites of interaction
by ~1.91 nm per domain31, which is unfolding event occurs. Such confor- between the A9 and G strands and the A
close to the expected difference (5 3 mational changes prior to unfolding and B strands are shown on an expanded
0.38 nm 5 1.90 nm). Insertion of glycine might also be important in regulating scale at the top and bottom, respectively.
residues between the I27 folds does not protein–protein interactions. A steered Amino acids, indicated by the single-
letter code, are shown in the boxes and
alter the unfolding interval because molecular dynamics simulation of forced
the hydrogen bonds between amino
these sections are fully stretched before unfolding of a FN-III domain suggests acids are indicated by the lines.
unfolding occurs31. Furthermore, as the that deformation of the integrin-binding
model implies, the final unfolding step ‘RGD’ motif occurs during an early stage
occurs as a single event. If there were of domain extension33. The application The energetics of force-induced unfolding
bonds positioned deeper within the fold of force might thereby cause a confor- and refolding
that were approximately as strong as mational change leading to the detach- Current models describe the energy
the A9–G bond, the unfolding of each do- ment of a bound integrin molecule from landscape for a folding protein as being
main would give multiple force peaks a FN-III domain. Thus, the RGD motif of similar to a funnel35–37. At the top of this
and the unfolding of multiple domains FN-III might act as a mechanosensitive funnel, proteins exist in a highly disor-
would be likely to occur in irregular, switch that controls the interaction dered, unfolded state of high energy and
interspersed steps. between cell-adhesion molecules. high entropy. Proteins are driven to as-
Despite the similarity in the chemical AFM refolding experiments have also sume progressively more ordered, lower
stability, spectrin domains unfold at identified misfolding of I27 domains34. energy conformations, until the native
much lower forces (25–35 pN)22 than Extension of refolded I278 rarely (2%) structure, with the lowest entropy and
do those of titin19 (150–300 pN) and resulted in force–extension curves with energy, is formed. The force-induced ex-
tenascin20 (a mean of 137 pN). Modelling peaks missing between apparently nor- tension of a protein, however, has added
of the forces involved in spectrin exten- mal unfolding events (‘skips’). The inter- implications. Under mechanical stress,
sion suggests that the unfolding dis- val between the peaks before and after the I27 domain is converted from its na-
tance for a spectrin a-helix is 1.5 nm, the skip corresponds to the size of two tive conformation (N) to a condensed
which is sixfold greater than that for I27 domains plus the number of amino denatured state (CD), in which it is
titin I27 (0.25 nm)27. Because unfolding acids between I27 folds. Skips therefore coiled but not folded (Fig. 6a). These
probability is exponentially dependent appear to represent misfolding events in conformations correspond to the en-
on the product of force and unfolding which the A strand of one domain inter- ergy states at the bottom and the top of
distance (see Fig. 1), this difference in acts with the G strand of the adjacent the ‘unfolding’ funnel. A third state, the
unfolding distance might explain the ob- domain, thereby creating a much larger extended denatured state (ED), occurs
served difference in mechanical stabil- fold that nevertheless has a stability only during mechanically induced un-
ity. The greater unfolding distance for similar to that of a native I27 fold. folding. A different energy landscape is
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REVIEWS TIBS 24 – OCTOBER 1999
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