Regeneration Studies of an Analog of Ribonuclease A Missing Disulfide Bonds 65−72 and 40−95

Abstract

Mutants of bovine pancreatic ribonuclease A (RNase A) that contain four of the eight cysteine residues found in the wild-type protein were prepared. Cysteine residues 40, 65, 72, and 95 were replaced by serine to form [C40S,C65S,C72S,C95S] RNase A or by alanine to form [C40A,C65A,C72A,C95A] RNase A, which contain the following four cysteine residues: 26, 58, 84, and 110. The substitutions resulted in deletion of wild-type disulfide bonds, 65-72 and 40-95. These mutants were prepared to investigate interactions that may be important for the folding and unfolding of the wild-type protein. The mutant protein was expressed and purified in an unfolded sulfonated form. Upon regeneration of the native form from the reduced mutant with DTTox, all three of the possible two-disulfide pairings, including the native one, formed. One-dimensional 1H NMR spectra demonstrated that the conformations of these three species are similar and are predominantly disordered; however, there is evidence of local structure in the vicinity of one histidine residue. It was also shown that disulfide pairing is not completely random and that both entropic factors and enthalpic interactions contribute to the formation of the native-disulfide bonds. The presence of more than a statistical population of native-disulfide pairings indicates that specific local interactions present in the reduced protein direct the preferential formation of native-disulfide bonds in the two-disulfide mutant.

Development of a Novel Method To Populate Native Disulfide-Bonded Intermediates for Structural Characterization of Proteins: Implications for the Mechanism of Oxidative Folding of RNase A†

“…In a previous different approach, mutant analogues of only one of these native two-disulfide-bonded intermediates were prepared . By contrast, in our study, all six possible native two-disulfide-bonded species were examined without replacing the four cysteines by alanines or serines.…”

Regeneration of Three-Disulfide Mutants of Bovine Pancreatic Ribonuclease A Missing the 65−72 Disulfide Bond: Characterization of a Minor Folding Pathway of Ribonuclease A and Kinetic Roles of Cys65 and Cys72

“…The two residues, used to replace cysteines in [C65S,C72S] and [C65A,C72A] for the regeneration experiments, were selected because serine and alanine are regarded as good substitutes for cysteine in protein refolding studies (34,35). However, it is known that serine and alanine mutants frequently show somewhat different refolding properties (14,36) for reasons that are not clear. Therefore, for a general comparison of mutants and wild-type RNase A in disulfidecoupled regeneration processes, we compared the regeneration kinetics of [C65S,C72S] and [C65A,C72A].…”

“…Recent studies in our 1 Abbreviations: RNase A, bovine pancreatic ribonuclease A; DTT ox , oxidized dithiothreitol; DTT red , DL-dithiothreitol; AEMTS, 2-aminoethylmethanethiosulfonate [(NH2)C2H4SSO2CH3]; des-[65-72], wild-type RNase A in which the 65-72 disulfide bond is reduced; des- , wild-type RNase A in which the 40-95 disulfide bond is reduced; HPLC, high-performance liquid chromatography; NMR, nuclear magnetic resonance; [C65S,C72S] and [C65A,C72A], the three-disulfide mutants of RNase A in which cysteines 65 and 72 have been replaced by serines and alanines, respectively; [C40A,C95A], the three disulfide mutant of RNase A in which cysteines 40 and 95 have been replaced by alanines; RP-HPLC, reverse-phase high-performance liquid chromatography; MALDI-TOF, matrix assisted laser desorption ionization-time-of-flight; Tris, tris(hydroxymethyl)aminomethane; EDTA, ethylenediaminetetraacetic acid; GdnSCN, guanidine thiocyanate; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; BPTI, bovine pancreatic trypsin inhibitor. laboratory involve DTT ox /DTT red -coupled regeneration experiments using AEMTS as a thiol-blocking agent (11)(12)(13) and the use of mutant proteins (14,15). A study of the folding properties of mutants is of interest because it helps to identify the roles of the substituted amino acids in the folding of the wild-type protein, by comparing the results for the mutants and the wild-type protein obtained under similar conditions.…”

Kinetic and thermodynamic analysis of the conformational folding process of SS‐reduced bovine pancreatic ribonuclease A using a selenoxide reagent with high oxidizing ability

“…6 shows that the total amount of N and the des intermediates generated in phase II is not enough to explain the UV absorbance change at 287 nm, suggesting generation of some intermediates having weakly folded structures other than the four des intermediates. One of the candidates for such intermediates would be the 2S intermediate with C58-C110 and C26-C84 SS bonds [59], which would populate more at low temperatures. Nevertheless, those intermediates are difficult to be observed due probably to the low populations as well as the facile reduction to R under the reduction pulse conditions.…”