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 the major oxidative folding pathway of bovine pancreatic ribonuclease A (RNase 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 species was unfolded at low pH to inhibit any disulfide reshuffling and was refolded at higher pH, allowing both conformational folding and disulfide-reshuffling reactions to take place. As a result of this competition, 15-85% of des-[40-95], depending on the experimental conditions, undergoes intramolecular disulfide-reshuffling reactions. That portion of the des-[40-95] population which has native isomers of essential proline residues appears to fold faster than the disulfide reaction can occur. However, when the folding is retarded, conceivably by the presence of non-native isomers of essential proline residues, des-[40-95] may reshuffle before completing the conformational folding process. These results enable us to distinguish among current models for the critical structure-forming step in oxidative folding and reveal a new model for coupling proline isomerization to disulfide-bond formation. These experiments also demonstrate that the reshuffling-folding competition assay is a useful tool for detecting structured populations in conformational folding intermediates.
Saito
K
K
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Welker
E
E
Scheraga
H A
HA
D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013696:Temperature
D004220:Disulfides; D013438:Sulfhydryl Compounds; D012259:Ribonuclease, Pancreatic