Kinetic role of a meta-stable native-like two-disulphide species in the folding transition of bovine pancreatic trypsin inhibitor.

The properties have been determined of a recently identified form of bovine pancreatic trypsin inhibitor, with only two of the three disulphide bonds of the native protein, but possessing a native-like conformation. The kinetics of refolding of the reduced inhibitor were re-measured to elucidate the kinetic role in folding of this two-disulphide species; it is formed both directly from a minor one-disulphide intermediate and by rearrangement of the other two-disulphide intermediates. It is not a productive intermediate because the Cys30 and Cys51 thiols are buried and unreactive. The previous kinetic analysis was extended by using both intra- and intermolecular disulphide reagents. Entirely consistent kinetic parameters for the rates of all the intramolecular steps of the pathway were obtained, and use of both types of reagents permits a detailed dissection of the kinetic pathway. In the process, the energetics of the folding transition were measured more thoroughly. The unique information available about the formation and stabilities of the disulphides during refolding of reduced bovine pancreatic trypsin inhibitor provides a useful description of the way in which numerous weak interactions within a protein co-operate to produce a stable folded conformation.