Conformational unfolding studies of three-disulfide mutants of bovine pancreatic ribonuclease A and the coupling of proline isomerization to disulfide redox reactions

The equilibrium stability and conformational unfolding kinetics of the [C40 A, C95A] and [C65S, C72S] mutants of bovine pancreatic ribonuclease A (RNas e A) have been studied. These mutants are analogues of two nativelike inter mediates, des[40-95] and des[65-72], whose formation is rate-limiting for o xidative folding and reductive unfolding at 25 degrees C and pH 8.0. Upon a ddition of guanidine hydrochloride, both mutants exhibit a fast conformatio nal unfolding phase when monitored by absorbance and fluorescence, as well as a slow phase detected only by fluorescence which corresponds to the isom erizations of Pro93 and Pro114. The amplitudes of the slow phase indicate t hat the two prolines, Pro93 and Pro114, are fully cis in the folded state o f the mutants and furthermore that the 40-95 disulfide bond is not responsi ble for the quenching of Tyr92 fluorescence observed in the slow unfolding phase, contrary to an earlier proposal [Rehage, A., and Schmid, F. X. (1982 ) Biochemistry 21, 1499-1505]. The ratio of the kinetic unfolding m double dagger value to the equilibrium m value indicates that the transition state for conformational unfolding in the mutants exposes little solvent-accessi ble area, as in the wild-type protein, indicating that the unfolding pathwa y is not dramatically altered by the reduction of the 40-95 or 65-72 disulf ide bond. The stabilities of the folded mutants are compared to that of wil d-type RNase A. These stabilities indicate that the reduction of des[40-95] to the 2S species is rate-limited by global conformational unfolding, wher eas that of des[65-72] is rate-limited by local conformational unfolding. T he isomerization of Pro93 may be rate-limiting for the reduction of the 40- 95 disulfide bond in the native protein and in the des[65-72] intermediate.