Abstract
Disulfide bonds are common to many extracellular proteins, where they serve to stabilize the native conformation. Indeed, the thiol/disulfide exchange mechanism is involved in important and complex biological processes. Much experimental and theoretical work seems to support the idea that the reaction proceeds through an uncomplicated SN2. While in gas phase a double minimum potential model describes the potential energy surface governing the chemical reaction, in solution phase only unimodal, concerted profiles without intermediates have been identified. However, the detailed shapes of the energy surfaces of these reactions are largely unknown, because the collisions with solvent control the trajectories of the molecules. The combination of molecular engineering techniques with single molecule force-clamp spectroscopy has made it possible to monitor the reduction of single disulfide bonds, allowing us to experimentally measure the bond elongation at the reaction transition state with sub-Angstrom resolution. Such an experimental approach provides an unprecedented experimental platform to directly probe the energy landscape of a simple chemical reaction in solution at the single bond level. By greatly expanding the range of pulling forces up to 1.5 nN, where covalent bonds are not yet broken, here we demonstrate that the disulfide bond cleavage by hydroxyl occurs through a double-barrier energy landscape. Whereas at low pulling forces (100-500pN) the reaction rate is limited by a first energy barrier exhibiting a distance to the transition state Δx ∼0.5 A, at higher forces (500-1500 pN) a second energy barrier exhibiting a shorter transition state of Δx ∼0.1 A becomes dominant. Our experimental approach allows us to probe regions of the energy landscape that were previously experimentally inaccessible, revealing signatures of unanticipated complexity.
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