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In-Depth Information
exchange as found by DFT calculations [110]. This result indicates that the force
dependence of the observed reaction kinetics is governed by the detected sub-Ångstrom
length changes between the two sulfur atoms at the reaction transition state. Such a
phenomenonmayapply tomanyother chemical reactions.Thus it seems that force-clamp
spectroscopy will be a powerful tool both to determine the effect of force on chemical
kinetics in general as well to directly probe the structure of chemical transition states.
13.7
Conclusions
Force-clamp spectroscopy has been highly successful in using mechanical force to
probe the physics and chemistry of proteins. In this chapter we have attempted to
demonstrate the robustness, reliability and ease of use of this technique to study
recombinant proteins. Force-clamp spectroscopy can be used to perturb the energy
landscape of a protein at a constant force along a well-de
ned reaction coordinate,
revealing
fine details of the transition states of folding, unfolding and even of protein
chemical reactions. These transition states range from nanometers down to the sub-
Ångstr
om scale, revealing the underlying physics involved in these processes. By
applying a constant force a number of force-dependent parameters can be obtained
with con
dence. While the force-clamp spectroscopy technique has developed
rapidly over the last few years, the instrumentation is still limited to a bandwidth
of a fewhundred hertz. Themost signi
cant development that we can expect to see in
the next few years is a force-clamp spectrometer with a megahertz bandwidth. Such a
development will permit a far more detailed examination of the energy landscape of a
protein, following their conformational dynamics with single bond resolution. Now
that the single-molecule
field is far from its infancy, the challenge is to develop
theoretical models of protein folding to incorporate force, as well as to consider the
interactions driving the microscopic mechanism of protein folding. The force-clamp
technique therefore unites
fields as diverse as protein biochemistry, statistical
mechanics far from equilibrium and the protein folding community.
€
Acknowledgments
We would like to express our gratitude to S. Garcia-Maynes and A. P. Wiita for helpful
discussions and critical reading of the chapter.
References
1 Oberhauser, A. and Carrión-V
azquez, M.
(2008) Mechanical Biochemistry of
Proteins One Molecule at a Time. J. Biol.
Chem.,
2 Ritort, F. (2006) Single-molecule
experiments in biological physics:
methods and applications. Journal of
Physics
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