Switching a Solvent Exposed Disulfide Bond into Methylene Thioacetal Reveals a New Hidden Path in the Folding Mechanism of BPTI and Enormously Enhances its Stability

Bovine pancreatic trypsin inhibitor (BPTI) is a 58 residues protein that is stabilized by three disulfide bonds at positions 5-55, 14-38 and 30-51. BPTI folding mechanism was extensively studied since it represents a folding model for many disulfide-rich proteins. The reduced protein folds via bifurcated pathway, where the reduced BPTI reach the native state via two intermediates: N’ that lacks the 5-55 disulfide and N* that lacks the 30-51. Formation of the fully oxidized native BPTI requires partial unfolding in a rate determining step that reduces the solvent exposed disulfide bridge 14-38 and rearrangement to form another intermediate N^SH_SH . Therefore, we hypothesized that replacing the 14-38 disulfide bond with highly stable methylene thioacetal, will cause the protein to be trapped at the two stable intermediates N’ and N*, preventing the formation of the native state (N). We performed this experiment and found that this is not the case! The methylene thioacetal substitution revealed a hidden pathway involving direct reaction between N* and the oxidizing reagent GSSG, to form the disulfide-mixed intermediate N*-SG, which directly form the N state. In addition, this substitution can turn to be a general strategy for enhancing protein stability while maintaining the protein structure and biological function.