Mechanochemistry: one bond at a time.

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((FΔx(r) - E(a))/k(B)T)[nucleophile]). From Arrhenius fits to the force dependency of the reduction rate, we measured the bond elongation parameter, Δx(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 Δx(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.