A localized specific interaction alters the unfolding pathways of structural homologues.
Reductive unfolding studies of proteins are designed to provide information about intramolecular 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 ribonuclease A, and through analysis of temperature 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 ribonuclease A and its structural homologue 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-containing loop region of ribonuclease A results in the localized modulation of protein dynamics that, in turn, enhances the susceptibility of the disulfide bond to reduction leading to an alteration in the reductive unfolding behavior of the homologues. These results have important implications for folding studies involving topological determinants to obtain folding/unfolding rates and pathways, for protein structure-function prediction through fold recognition, and for predicting proteolytic cleavage sites.
Xu
Guoqiang
G
Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Narayan
Mahesh
M
Kurinov
Igor
I
Ripoll
Daniel R
DR
Welker
Ervin
E
Khalili
Mey
M
Ealick
Steven E
SE
Scheraga
Harold A
HA
D000818:Animals; D002417:Cattle; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
D011994:Recombinant Proteins; D012260:Ribonucleases; D012259:Ribonuclease, Pancreatic; C058703:ranpirnase