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Liang J, Fernandez JM: Mechanochemistry: One Bond at a Time. ACS Nano. 2009 Jul 2.
Single-molecule force-clamp spectroscopy offers a novel platform for mechanically denaturing proteins by applying a constant force to a polyprotein. A powerful emerging application of the technique is that, by introducing a disulfide bond in each protein module, the chemical kinetics of disulfide bond cleavage under different stretching forces can be probed at the single-bond level. Even at forces much lower than that which can rupture the chemical bond, the breaking of the S-S bond at the presence of various chemical reducing agents is significantly accelerated. Our previous work demonstrated that the rate of thiol/disulfide exchange reaction is force-dependent and well-described by an Arrhenius term of the form r = A (exp ((FDeltax (r) - E (a))/k (B) T)[nucleophile]). From Arrhenius fits to the force dependency of the reduction rate, we measured the bond elongation parameter, Deltax (r), along the reaction coordinate to the transition state of the S (N) 2 reaction cleaved by different nucleophiles and enzymes, never before observed by any other technique. For S-S cleavage by various reducing agents, obtaining the Deltax (r) value can help depicting the energy landscapes and elucidating the mechanisms of the reactions at the single-molecule level. Small nucleophiles, such as 1,4-dl-dithiothreitol (DTT), tris (2-carboxyethyl) phosphine (TCEP), and l-cysteine, react with the S-S bond with monotonically increasing rates under the applied force, while thioredoxin enzymes exhibit both stretching-favored and -resistant reaction-rate regimes. These measurements demonstrate the power of the single-molecule force-clamp spectroscopy approach in providing unprecedented access to chemical reactions. |
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