Refolding of bovine pancreatic trypsin inhibitor via non-native disulphide intermediates.

The disulphide folding pathway of bovine pancreatic trypsin inhibitor (BPTI), especially at the two-disulphide stage, has been dissected by replacing one or two particular cysteine residues by serine. This restricts which disulphide species are possible, and the observed kinetics of disulphide-coupled folding reveal the roles of the remaining species. The results obtained confirm the kinetic roles in the original BPTI pathway of the two specific two-disulphide intermediates with non-native second disulphide bonds, (30-51, 5-14) and (30-51, 5-38). Moreover, the rates of folding through each of these intermediates are shown to account quantitatively for the rate of folding of the normal protein; therefore, essentially all the molecules refold through these two particular intermediates. They are amongst the most productive on the folding pathway, and their roles are readily explicable on the basis of their conformations.