The molecular mechanisms underlying thiol-based redox control are poorly defined. Disulfide bonds between Cys residues are commonly thought to confer extra rigidity and stability to their resident protein, forming a type of proteinaceous spot weld. Redox biologists have been redefining the role of disulfides over the last 30-40 years. Disulfides are now known to form in the cytosol under conditions of oxidative stress. Isomerization of extracellular disulfides is also emerging as an important regulator of protein function. The current paradigm is that the disulfide proteome consists of two subproteomes: a structural group and a redox-sensitive group. The redox-sensitive group is less stable and often associated with regions of stress in protein structures. Some characterized redox-active disulfides are the helical CXXC motif, often associated with thioredoxin-fold proteins; and forbidden disulfides, a group of metastable disulfides that disobey elucidated rules of protein stereochemistry. Here we discuss the role of redox-active disulfides as switches in proteins.

Disulfides are conventionally viewed as structurally stabilizing elements in proteins but emerging evidence suggests two disulfide subproteomes exist. One group mediates the well known role of structural stabilization. A second redox-active group are best known for their catalytic functions but are increasingly being recognized for their roles in regulation of protein function. Redox-active disulfides are, by their very nature, more susceptible to reduction than structural disulfides; and conversely, the Cys pairs that form them are more susceptible to oxidation. In this study, we searched for potentially redox-active Cys Pairs by scanning the Protein Data Bank for structures of proteins in alternate redox states. The PDB contains over 1134 unique redox pairs of proteins, many of which exhibit conformational differences between alternate redox states. Several classes of structural changes were observed, proteins that exhibit: disulfide oxidation following expulsion of metals such as zinc; major reorganisation of the polypeptide backbone in association with disulfide redox-activity; order/disorder transitions; and changes in quaternary structure. Based on evidence gathered supporting disulfide redox activity, we propose disulfides present in alternate redox states are likely to have physiologically relevant redox activity.

Disulfide torsional energy, a good predictor of disulfide redox potential in proteins, may be estimated by interpolation on a potential energy surface (PES) describing the twisting of diethyl disulfide through its three central dihedral angles. Here we update PES calculations at the M05-2X level of theory with the 6-31G(d) basis set. Although the surface shows no qualitative differences from an earlier MP2(full) PES, energy differences greater than 1 kJ mol–1 were seen for conformations with χ2 between –60° and 30°, or with χ3 below 60° or above 130°. This is particularly significant for highly strained disulfides that are likely to be spontaneously reduced by mechanical means. In benchmarking against the high-level G3X method, M05-2X showed significantly reduced root mean squared deviation compared with MP2(full) (1.0 versus 2.0 kJ mol–1 respectively). Results are incorporated into a web application that calculates relative torsional energies from disulfide dihedral angles (http://www.sbinf.org/applications/pes.html).