Direct observation of disulfide isomerization in a single protein

Abstract

Photochemical uncaging techniques use light to release active molecules from otherwise inert compounds. Here we expand this class of techniques by demonstrating the mechanical uncaging of a reactive species within a single protein. We proved this novel technique by capturing the regiospecific reaction between a thiol and a vicinal disulfide bond. We designed a protein that includes a caged cysteine and a buried disulfide. The mechanical unfolding of this protein in the presence of an external nucleophile frees the single reactive cysteine residue, which now can cleave the target disulfide via a nucleophilic attack on either one of its two sulfur atoms. This produces two different and competing reaction pathways. We used single-molecule force spectroscopy to monitor the cleavage of the disulfides, which extends the polypeptide by a magnitude unambiguously associated with each reaction pathway. This allowed us to measure, for the first time, the kinetics of disulfide-bond isomerization in a protein. Multiple redox reaction pathways exist in proteins containing several cysteines. A technique termed mechanical uncaging is now demonstrated, allowing the release of a single reactive cysteine within a protein and the unequivocal observation of subsequent thiol/disulfide exchanges. Mechanical uncaging of reactive groups is useful for studying chemical kinetics in a synchronized manner.