Effect of mutation of proline 93 on redox unfolding/folding of bovine pancreatic ribonuclease A.
Both the reductive unfolding and oxidative regeneration of a P93A mutant and wild-type RNase A have been studied at 15 degrees C and pH 8.0. The rate of reduction of the 40--95 disulfide bond is accelerated about 120-fold by the P93A mutation, while the reduction of the 65--72 disulfide bond is not accelerated by this mutation (within the experimental error). Moreover, the reduction of native P93A to des[40--95] is about 10 times faster than the further reduction of the same des[40--95] species. These results demonstrate that the reduction of the mutant proceeds through a local unfolding event and provides strong support for our model in which the reduction of wild-type RNase A to the des species proceeds through two independent local conformational unfolding events. The oxidative regeneration rate of the P93A mutant is comparable to that of wild-type RNase A, suggesting that a cis 92--93 peptide group that is present in native wild-type RNase A and in native des[40--95], is not obligatory for the formation of the third (final) native disulfide bond of des[40--95] by reshuffling from an unstructured 3S precursor. Thus, the trans to cis isomerization of the Tyr92-Pro93 peptide group during the regeneration of wild-type RNase A may occur after the formation of the third native disulfide bond.
Cao
A
A
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Welker
E
E
Scheraga
H A
HA
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004220:Disulfides / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D017354:Point Mutation; D011392:Proline / Q000235:genetics*; D011487:Protein Conformation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000235:genetics* / Q000378:metabolism*
D004220:Disulfides; D011392:Proline; D012259:Ribonuclease, Pancreatic; D000409:Alanine