Single-molecule force clamp spectroscopy is an atomic force microscopy (AFM)-based technique in which single molecules (e.g. polyproteins) are mechanically stretched under constant force while their elastic response is recorded at real time. An emerging application of the technique relies on its ability for exploring the chemical kinetics of disulfide bond cleavage under different stretching forces at single-bond level. Recently, we showed that by carrying out the force-dependent disulfide bond reduction experiments at a series of well-controlled temperatures, the pre-exponential factor A and the activation energy Ea could be measured independently. For instance, the reaction rate of the disulfide bond cleavage by nucleophilic attack of tris(2-carboxyethyl)phosphine (TCEP) increases monotonically with temperature and, A is measured to be at the order of 107 M-1s-1, far lower than that predicted by the transition-state theory, in which A is given by kBT/h and around 1012 M-1s-1 at room temperature.

Disulfide bond reduction kinetics measured by single-molecule force clamp spectroscopy

243rd National Meeting (2012)

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