Fig. 2.
Mechanical force can induce thermodynamically non-spontaneous disulfide bond cleavage. a Schematic of the single-molecule force-clamp experiment, whereby a single (I27E24C-K55C)8 polyprotein is stretched by an AFM cantilever. b In the mechanochemical assay, mechanical force unfolds the protein (gray region) up to the mechanically rigid disulfide bond (formed between cysteine 24 and 55), which becomes solvent exposed. The presence of a nucleophile triggers disulfide cleavage, fingerprinted by the 10 nm steps that correspond to the extension of the protein amino acids that were trapped behind the disulfide bond (red region). The resulting reduced and mechanically unfolded protein harbors a mixed disulfide and a reduced protein thiolate. c Individual reduction trajectory demonstrating that the protein disulfide bond can be cleaved by 2 mM cysteine-methyl-ester (black asterisks mark the reduction of each individual disulfide bond under force, fingerprinted by a stepwise increase in the protein length of ~10 nm). d Disulfide bond rupture kinetics at F = 350 pN for the thermodynamically largely disfavored disulfide bond reduction by Cys-ME (red) and the marginally un-favored reaction in the presence of penicillamine (dark orange), compared to that of L-cysteine (orange). All compounds were kept at a constant deprotonated concentration [S−] = 2 mM. e The concentration-normalized rate of rupture for all compounds at a constant F = 350 pN correlates with the electrostatic partial charge localized on the nucleophilic sulfur. (Inset: charge distribution plot of Cys-ME, from red (negative), to green (~0) to blue (positive)) (From left to right n = 13, 24, 23, 17, 35, 23, 34, and 17, where n = number of individual trajectories). f The force-dependent reduction kinetics for NAC, thioglycerol, mesna, and glutathione demonstrates that all compounds exhibit a similar force-sensitivity yet different nucleophilicity; (From left to right: nNAC = 35, 17, 42, and 24; nThioglycerol = 19, 35, 21, and 27; nMesna = 19, 35, 21, and 27; nGlutathione = 37, 17, 41, and 16; error bars: s.d)