A single-molecule assay to directly identify solvent-accessible disulfide bonds and probe their effect on protein folding.
J Am Chem Soc
Disulfide bonds are ubiquitous in proteins. According to a recent survey, there are 97 741 disulfide bonds in 121 779 protein structures available in the Protein Data Bank (PDB). Native as well as engineered disulfide bonds have been shown to control the stability and function of proteins. The redox state of protein disulfide bonds in vivo, governing protein stability and function, depends on the disulfide bond accessibility to the surrounding reducing agent molecules in the intracellular environment as well as those present on the cell surface. While structural techniques such as X-ray crystallography can directly identify protein disulfide bonds, they only provide information on static accessibility of the disulfide bonds to the surroundings. However, proteins are known to be dynamic in nature. In the case of proteins for which structures are not available, indirect methods, such as measuring the changes in protein mobility by SDS-PAGE in different redox conditions, analyzing digested protein fragments by mass spectrometry and quantifying the thiols of reduced disulfide bonds by the Ellman’s reagent, have been implemented to identify protein disulfide bonds. While these assays are useful for identifying the presence of disulfide bonds in general, they often require protein denaturation and disulfide cleavage and are typically unable to identify intramolecular disulfide bonds in their oxidized state and directly measure their solvent accessibility. Here, we present single-molecule force spectroscopy as a tool to directly identify intramolecular protein disulfide bonds in their oxidized state, measure their accessibility to small-molecule reducing agents in the bathing solution, and probe the differential folding kinetics of reduced and oxidized proteins at the single-molecule level.