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

Brian P. English, E. Welker, Mahesh Narayan, Harold A. Scheraga

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6 Citations (Scopus)

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 nativelike structured three-disulfide-bonded intermediates populated between 8 and 25 °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 nativelike structured des species whose isolation had thus far not been possible.

Original languageEnglish
Pages (from-to)4995-4999
Number of pages5
JournalJournal of the American Chemical Society
Volume124
Issue number18
DOIs
Publication statusPublished - May 8 2002

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Pancreatic Ribonuclease
Disulfides
Proteins
Protein Folding
Oxidation
Ribonucleases

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

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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 nativelike structured three-disulfide-bonded intermediates populated between 8 and 25 °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 nativelike structured des species whose isolation had thus far not been possible.",
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AU - English, Brian P.

AU - Welker, E.

AU - Narayan, Mahesh

AU - Scheraga, Harold A.

PY - 2002/5/8

Y1 - 2002/5/8

N2 - 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 nativelike structured three-disulfide-bonded intermediates populated between 8 and 25 °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 nativelike structured des species whose isolation had thus far not been possible.

AB - 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 nativelike structured three-disulfide-bonded intermediates populated between 8 and 25 °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 nativelike structured des species whose isolation had thus far not been possible.

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