Disulfide bonds and protein folding

The applications of disulfide-bond chemistry to studies of protein folding, structure, and stability are reviewed and illustrated with bovine pancreat ic ribonuclease A (RNase A). After surveying the general properties and adv antages of disulfide-bond studies, we illustrate the mechanism of reductive unfolding with RNase A, and discuss its application to probing structural fluctuations in folded proteins. The oxidative folding of RNase A is then d escribed, focusing on the role of structure formation in the regeneration o f the native disulfide bonds. The development of structure and conformation al order in the disulfide intermediates during oxidative folding is charact erized. Partially folded disulfide species are not observed, indicating tha t disulfide-coupled folding is highly cooperative. Contrary to the predicti ons of "rugged funnel" models of protein folding, misfolded disulfide speci es are also not observed despite the potentially stabilizing effect of many nonnative disulfide bonds. The mechanism of regenerating the native disulf ide bonds suggests an analogous scenario for conformational folding. Finall y, engineered covalent crosslinks may be used to assay for the association of protein segments in the folding transition state, as illustrated with RN ase A.