MODULATING THE REDUCTIVE UNFOLDING PATHWAY OF RNASE A

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Reductive unfolding studies of proteins are designed to provide information about intramol. interactions that govern the formation (and stabilization) of the native state and about folding/unfolding pathways. By mutating Tyr92 to G, A, or L in the model protein, bovine pancreatic RNase A, and through analysis of temp. factors and molecular dynamics simulations of the crystal structures of these mutants, it is demonstrated that the markedly different reductive unfolding rates and pathways of RNase A and its structural homolog onconase can be attributed to a single, localized, ring-stacking interaction between Tyr92 and Pro93 in the bovine variant. The fortuitous location of this specific stabilizing interaction in a disulfide-bond-contg. loop region of RNase A results in the localized modulation of protein dynamics that, in turn, enhances the susceptibility of the disulfide bond to redn. leading to an alteration in the reductive unfolding behavior of the homologues. These results have important implications for folding studies involving topol. determinants to obtain folding/unfolding rates and pathways, for protein structure-function prediction through fold recognition, and for predicting proteolytic cleavage sites. Three structures were deposited in the PDB (1YMR, 1YMW, and 1YMN) and the work was published in JACS.