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 [C40A, C95A] and [C65S, C72S] mutants of bovine pancreatic ribonuclease A (RNase A) have been studied. These mutants are analogues of two nativelike intermediates, des[40-95] and des[65-72], whose formation is rate-limiting for oxidative folding and reductive unfolding at 25 degrees C and pH 8.0. Upon addition of guanidine hydrochloride, both mutants exhibit a fast conformational unfolding phase when monitored by absorbance and fluorescence, as well as a slow phase detected only by fluorescence which corresponds to the isomerizations of Pro93 and Pro114. The amplitudes of the slow phase indicate that the two prolines, Pro93 and Pro114, are fully cis in the folded state of the mutants and furthermore that the 40-95 disulfide bond is not responsible 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 value to the equilibrium m value indicates that the transition state for conformational unfolding in the mutants exposes little solvent-accessible area, as in the wild-type protein, indicating that the unfolding pathway is not dramatically altered by the reduction of the 40-95 or 65-72 disulfide bond. The stabilities of the folded mutants are compared to that of wild-type RNase A. These stabilities indicate that the reduction of des[40-95] to the 2S species is rate-limited by global conformational unfolding, whereas that of des[65-72] is rate-limited by local conformational unfolding. The 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.
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
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Iwaoka
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Wedemeyer
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Scheraga
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000235:genetics*; D004220:Disulfides / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011392:Proline / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics*; D012694:Serine / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D004220:Disulfides; D020033:Protein Isoforms; D012694:Serine; D011392:Proline; D012259:Ribonuclease, Pancreatic; D003545:Cysteine; D000409:Alanine