A method for determining the kinetic fate of structured disulfide species (
i.e., whether they are preferentially oxidized or reshuffle back to an unst
ructured disulfide species) is introduced. The method relies on the sensiti
vity of unstructured disulfide species to low concentrations of reducing ag
ents. Because a structured des species that preferentially reshuffles gener
ally first rearranges to an unstructured species, a small concentration of
reduced DTT (e.g., 260 muM) suffices to distinguish on-pathway intermediate
s from dead-end species. We apply this method to the oxidative folding of b
ovine pancreatic ribonuclease A (RNase A) and show that des[40-95] and des[
65-72] are productive intermediates, whereas des[26-84] and des[58-110] are
metastable dead-end species that preferentially reshuffle. The key factor
in determining the kinetic fate of these des species is the relative access
ibility of both their thiol groups and disulfide bonds. Productive intermed
iates tend to be disulfide-secure, meaning that their structural fluctuatio
ns preferentially expose their thiol groups, while keeping their disulfide
bonds buried. By contrast, dead-end species tend to be disulfide-insecure,
in that their structural fluctuations expose their disulfide bonds in conce
rt with their thiol groups. This distinction leads to four generic types of
oxidative folding pathways. We combine these results with those of earlier
studies to suggest a general three-stage model of oxidative folding of RNa
se A and other single-domain proteins with multiple disulfide bonds.