Mechanochemistry: One bond at a time

摘要

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 _BT)[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_N2 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.