Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques
Proc Natl Acad Sci U S A
The mechanism by which mechanical force regulates the kinetics of a chemical reaction is unknown. Here, we use single-molecule force–clamp spectroscopy and protein engineering to study the effect of force on the kinetics of thiol/disulfide exchange. Reduction of disulfide bonds through the thiol/disulfide exchange chemical reaction is crucial in regulating protein function and is known to occur in mechanically stressed proteins. We apply a constant stretching force to single engineered disulfide bonds and measure their rate of reduction by DTT. Although the reduction rate is linearly dependent on the concentration of DTT, it is exponentially dependent on the applied force, increasing 10-fold over a 300-pN range. This result predicts that the disulfide bond lengthens by 0.34 Å at the transition state of the thiol/disulfide exchange reaction. Our work at the single bond level directly demonstrates that thiol/disulfide exchange in proteins is a force-dependent chemical reaction. Our findings suggest that mechanical force plays a role in disulfide reduction in vivo, a property that has never been explored by traditional biochemistry. Furthermore, our work also indicates that the kinetics of any chemical reaction that results in bond lengthening will be force-dependent.