Clamps force

Oberhauser AF, Hansma PK, Carrion-Vazquez M, Fernandez JM. Stepwise unfolding of titin under force-clamp atomic force microscopy. Proc Natl Acad Sci USA 2001 98 468-472. 

Visscher, K., M.J. Schnitzer, and S.M. Block. 1999. Single kinesin molecules studied with a molecular force clamp. Nature 400 184-189. 

Fig. 17.3. Step size histograms obtained from force-clamp traces measnred from (a) single 127 proteins where all observed steps are considered or from (b) 1278 polyproteins where only staircases of three or more identical steps are considered [16]. The figure shows that the polyprotein fingerprint (b) weeds out spurious interactions (a)...

Force-Clamp Spectroscopy Measures the Distance to the Transition State Ax... 

Fig. 17.5c, solid line) to determine the values of AG and Ax. These force-clamp experiments show that the distance to the transition state of unfolding of the 127 protein, Ax = 2.4 A, which is the size of a water molecule [24]. In addition, the value of the unfolding rate extrapolated to zero force measures the size of the activation energy barrier AG. However, AG obtained from the Arrhenius fits depends on the value used for the attempt frequency A (Fig. 17.4), which needs to be measured independently for each reaction [24]. By contrast, the value of Ax is measured from the slope of the Arrhenius plot, and thus is independent of the value of the attempt frequency A. 

Perhaps the most striking use of force-clamp spectroscopy so far has been in the study of the effect of force on a chemical reaction [13-15]. While mortars and pestles have been used for thousands of years to catalyze chemical reactions, it had never been possible to examine a molecule undergoing a chemical reaction when placed under a calibrated and vectorially defined force. This has now been achieved by combining protein engineering with force-clamp spectroscopy. Our basic strategy is shown in Fig. 17.7. 

In order to study the kinetics of disulfide bond reduction as a function of the pulling force, we utilize a double pulse protocol in force clamp. With a first pulse, we unfold the unsequestered region of the (I27s-s)8 modules in the polyprotein, exposing the disulfide bonds to the solution. With a second (test) pulse, we track the rate of reduction of the exposed disulfides at various pulling forces in the presence of various reducing agents. 

Force-Clamp Spectroscopy of Single Proteins Reduction events 13.5 nm... 

The sub-Angstrom resolution of the transition state d3mamics of a chemical reaction obtained using force-clamp techniques makes a novel contribution to our understanding of protein-based chemical reactions. These experiments, combined with quantum chemical predictions of transition state structure, hold promise for developing a quantitative view of enz3mie catalysis and other protein-based chemical reactions, at a resolution currently unattainable by any other means. 

W.J. Greenleaf, M.T. Woodside, E.A. Abbondanzieri, S.M. Block, Passive all-optical force clamp for high-resolution laser trapping. Phys. Rev. Lett. 95, 208102 (2005)... 

Block SM, Asbury CL, Shaevitz JW, Lang MJ. 2003. Probing the kinesin reaction cycle with a 2D optical force clamp. Proceedings of the National Academy of Sciences of USA 100 2351. 

Schlierf M, Li H, Fernandez JM (2004) The uttfolding kinetics of ubiquitin captured with single-molecule force-clamp techniques. Proc Natl Acad Sci USA 101 7299-7304... 

Fernandez JM, Li H (2004) Force-clamp spectroscopy monitors the folding trajectory of a single protein. Science 303 1674—1678... 

Given that our model system was smaller than the protein employed in the force-clamp AI experiments, the very first thing that needs to be proven is that the observed force-induced conformational rearrangement in the diethyl disulfide molecule can also take place in the protein and, thus, that the mechanism extracted from this model is responsible for the experimental findings reported for the protein. Force-field molecular dynamics simulations carried out on the same system used in... 

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