Effects of Tyrosine Mutations on the Conformational and Oxidative Folding of Ribonuclease A: A Comparative Study

Abstract

Ribonuclease A (RNase A) undergoes more rapid conformational folding with its disulfide bonds intact than during oxidative folding from its reduced form. In this study, the effect of the mutants Y92G, Y92A, and Y92L on both the conformational and oxidative folding pathways was examined in order to determine the role of native interactions in different types of conformational searches to obtain the biologically active structure of a protein. These mutations did not affect the overall conformational folding pathway of RNase A. However, in the mutants Y92G and Y92A, a key structured disulfide-bonded species, des-[65-72], involved in the oxidative folding pathway of RNase A, was destabilized. These results demonstrate the importance of native interactions in the folding process, namely, protection of a native (40-95) disulfide bond by a nearby tyrosyl-prolyl stacking interaction, when disulfide bonds are allowed to undergo SH/S-S reshuffling.

Insight into the Initial Stages of the Folding Process in Onconase Revealed by UNRES

“…By allowing reshuffling to occur in the folding process, the formation of a native-like structure can take much longer than if the native disulfide bonds are intact during folding and favorable interactions play an even more important role in the formation of the native structure. 8 This folding process occurs in the order of seconds compared to milliseconds as observed with disulfide-bond intact conformational folding. 9 In addition, if the reshuffling reactions are halted, the formation of native structure is arrested.…”

“…As folding proceeds, favorable interactions are formed and re-formed concomitantly with cysteines forming and re-forming disulfide bonds, also termed reshuffling, until the native structure with native disulfide connectivity is achieved. By allowing reshuffling to occur in the folding process, the formation of a native-like structure can take much longer than if the native disulfide bonds are intact during folding and favorable interactions play an even more important role in the formation of the native structure . This folding process occurs in the order of seconds compared to milliseconds as observed with disulfide-bond intact conformational folding .…”