Role of the [65-72] disulfide bond in oxidative folding of bovine pancreatic ribonuclease A.
To assess the role of the [65-72] disulfide bond in the oxidative folding of RNase A, use has been made of [C65S, C72S], a three-disulfide-containing mutant of RNase A which regenerates from its two-disulfide precursor in an oxidation and conformational folding-coupled rate-determining step. The distribution of disulfide bonds in the one-disulfide-containing ensemble of this mutant has been characterized. In general, the disulfide-bond distribution in its 1S ensemble agrees relatively well with the corresponding distribution in wt-RNase A and with distributions based on calculations of loop entropy, except for the absence of the [65-72] disulfide bond. There is no bias (over the entropic influence) for the three native disulfide bonds, [26-84], [40-95], and [58-110]. Previous oxidative folding results for wt-RNase A indicated the predominance of the des [40-95] intermediate over des [65-72] after the rate-determining step in the regeneration process. Considering that there is no preferential distribution of disulfides in the 1S ensemble of [C65S, C72S], in contrast to the preferential population of the [65-72] disulfide bond in wt-RNase A, these results indicate a critical role for the [65-72] disulfide bond in the regeneration of wt-RNase A. Furthermore, analysis of the disulfide distribution of the 1S intermediates of [C65S, C72S] compared to that of wt-RNase A lends support for a physicochemical basis for the previously observed slow folding rate of this mutant, compared to its analogue (des [65-72]) of wt-RNase A.
D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange / Q000379:methods; D002918:Chymotrypsin / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D007700:Kinetics; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D014357:Trypsin / Q000737:chemistry
D004220:Disulfides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012259:Ribonuclease, Pancreatic; D002918:Chymotrypsin; D014357:Trypsin