A kinetic explanation for the rearrangement pathway of BPTI folding.

Bovine pancreatic trypsin inhibitor (BPTI) does not fold by simple sequential formation of its native disulphide bonds. Instead, an initially formed intermediate, termed N', first rearranges to a more stable species in a slow process that requires substantial unfolding. We find that direct oxidation of N' is also inhibited by native structure which slows both the intermolecular step in oxidation--formation of a mixed disulphide bond with the oxidizing agent GSSG--as well as the subsequent intramolecular step. Folding does not occur appreciably by direct oxidation because the high GSSG concentrations required for efficient mixed disulphide formation cause N' to accumulate as a nonproductive, double-mixed disulphide species. The need to unfold previously acquired native structure, observed in the folding of BPTI, may be a common feature of disulphide-linked folding reactions.