EFFECTS OF AMINO-ACID REPLACEMENTS ON THE REDUCTIVE UNFOLDING KINETICS OF PANCREATIC TRYPSIN-INHIBITOR

In order to characterize the major transition states in the disulfide- coupled folding pathway of bovine pancreatic trypsin inhibitor (BPTI), the reductive unfolding kinetics of wild-type BPTI and 18 variants wi th single amino acid replacements were measured in the presence of var ying concentrations of dithiothreitol (DTTSHSH). As observed previousl y for the wild-type protein, unfolding of the mutant proteins was foun d to proceed through the formation of a native-like two-disulfide inte rmediate (N-SH(SH)), followed by either direct reduction of this inter mediate or intramolecular rearrangement to generate other two-disulfid e species that were then reduced further. From the dependence of the r ate of disappearance of N-SH(SH) on the concentration of DTTSHSH, the rate constants for the direct and rearrangement mechanisms were estima ted. All of the amino acid replacements examined were found to increas e both rate constants, with some mutants unfolding as much as 10 000-f old more rapidly than the wild-type protein. The two rate constants we re highly correlated by a linear free energy relationship, suggesting that the transition states for the direct and rearrangement mechanisms are very similar in their response to amino acid replacements. These results are consistent with a model in which the two transition states , which are also the major transition states for disulfide-coupled ref olding, are extensively unfolded.