title: 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.
abstract: RNase A, a model protein for oxidative folding studies, has four native disulfide bonds. The roles of des [40-95] and des [65-72], the two native-like structured three-disulfide-bonded intermediates populated between 8 and 25 degrees C during the oxidative folding of RNase A, are well characterized. Recent work focuses on both the formation of these structured disulfide intermediates from their unstructured precursors and on the subsequent oxidation of the structured species to form the native protein. The major obstacles in this work are the very low concentration of the precursor species and the difficulty of isolating some of the structured intermediates. Here, we demonstrate a novel method that enables the native disulfide-bonded intermediates to be populated and studied regardless of whether they have stable structure and/or are present at low concentrations during the oxidative folding or reductive unfolding process. The application of this method enabled us to populate and, in turn, study the key intermediates with two native disulfide bonds on the oxidative folding pathway of RNase A; it also facilitated the isolation of des [58-110] and des [26-84], the other two native-like structured des species whose isolation had thus far not been possible.
authors: Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
authors: Brian P
authors: BP
authors: English
authors: Ervin
authors: E
authors: Welker
authors: Mahesh
authors: M
authors: Narayan
authors: Harold A
authors: HA
authors: Scheraga
mesh_terms: D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000737:chemistry*; D007700:Kinetics; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*
chemical_list: D004220:Disulfides; D010720:Phosphines; D012259:Ribonuclease, Pancreatic; C080938:tris(2-carboxyethyl)phosphine