The Temperature Dependency of Disulfide Bond Reduction Events Measured by Single-molecule Force Clamp Spectroscopy

Abstract

An emerging application of single-molecule force clamp spectroscopy is the ability to explore the chemical kinetics of disulfide bond cleavage under different stretching forces at the single-bond level. Our previous work demonstrated that the rate of thiol/disulfide exchange reaction in proteins is force-dependent, and well described by an Arrhenius term of the form: r = A(exp((FΔxr-Ea)/kBT)[nucleophile]). From the force dependency of the reduction rate we could measure the bond elongation, Δxr (0.2-0.6 A), that occurs at the transition state of the SN2 reaction cleaved by different chemical reagents and enzymes, never before observed by any other technique. However, our estimates of Ea (53-60 kJ/mol) were uncertain and dependent on our choice of value for A (1012 M−1s−1). Here, we show that by carrying out the force-dependent disulfide bond reduction experiments at a series of well-controlled temperatures, A can be measured independently. We demonstrate that 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, which is 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. Factoring in the much lower value of A, Ea is calculated to be 35 kJ/mol, which is much lower than 58 kJ/mol that we had reported previously. For thioredoxin (Trx)-catalyzed disulfide bond cleavage, obtaining the A, Ea and Δxr values can help elucidating the reaction mechanisms and the role of temperature in the regulation of Trx activity, which is of special interest when comparing enzymes from different species. These measurements demonstrate the power of single-molecule force spectroscopy approach in providing unprecedented access to protein based chemical reactions.