Abstract
Bovine pancreatic trypsin inhibitor (BPTI) is a 58-residue protein that is stabilized by three disulfide bonds at positions 5-55, 14-38 and 30-51. Widely studied for about 50 years, BPTI represents a folding model for many disulfide-rich proteins. In the study described below, we replaced the solvent exposed 14-38 disulfide bond with a methylene thioacetal bridge in an attempt to arrest the folding pathway of the protein at its two well-known intermediates, N' and N*. The modified protein was expected to be unable to undergo the rate-determining step in the widely accepted BPTI folding mechanism: the opening of the 14-38 disulfide bond followed by rearrangements that leads to the native state, N. Surprisingly, instead of halting BPTI folding at N' and N*, we uncovered a hidden pathway involving a direct reaction between the N* intermediate and the oxidizing reagent glutathione (GSSG) to form the disulfide-mixed intermediate N*-SG, which spontaneously folds into N. On the other hand, N' was unable to fold into N. In addition, we found that the methylene thioacetal bridge enhances BPTI stability while fully maintaining its structure and biological function. These findings suggest a general strategy for enhancing protein stability without compromising on function or structure, suggesting potential applications for future therapeutic protein production.