Folding of a disulfide-bonded protein species with free thiol(s): Competition between conformational folding and disulfide reshuffling in an intermediate of bovine pancreatic ribonuclease A

The conformational folding of the nativelike intermediate des-[40-95] on th e major oxidative folding pathway of bovine pancreatic ribonuclease A (RNas e A) has been examined at various pHs and temperatures in the absence of a redox reagent. Des-[40-95] has three of the four disulfide bonds of native RNase A and lacks the bond between Cys40 and Cys95. This three-disulfide sp ecies was unfolded at low pH to inhibit any disulfide reshuffling and was r efolded at higher pH, allowing both conformational folding and disulfide-re shuffling reactions to take place. As a result of this competition, 15-85% of des-[40-95], depending on the experimental conditions, undergoes intramo lecular disulfide-reshuffling reactions. That portion of the des-[40-95] po pulation which has native isomers of essential proline residues appears to fold faster than the disulfide reaction can occur. However, when the foldin g is retarded, conceivably by the presence of non-native isomers of essenti al proline residues, des-[40-95] may reshuffle before completing the confor mational folding process. These results enable us to distinguish among curr ent models for the critical structure-forming step in oxidative folding and reveal a new model for coupling proline isomerization to disulfide-bond fo rmation. These experiments also demonstrate that the reshuffling-folding co mpetition assay is a useful tool for detecting structured populations in co nformational folding intermediates.