Structural determinants of oxidative folding in proteins.
Proc Natl Acad Sci U S A
; 98(5): 2312-6, 2001 Feb 27.
Article
em En
| MEDLINE
| ID: mdl-11226236
ABSTRACT
A method for determining the kinetic fate of structured disulfide species (i.e., whether they are preferentially oxidized or reshuffle back to an unstructured disulfide species) is introduced. The method relies on the sensitivity of unstructured disulfide species to low concentrations of reducing agents. Because a structured des species that preferentially reshuffles generally first rearranges to an unstructured species, a small concentration of reduced DTT (e.g., 260 microM) suffices to distinguish on-pathway intermediates from dead-end species. We apply this method to the oxidative folding of bovine pancreatic ribonuclease A (RNase A) and show that des[40-95] and des[65-72] are productive intermediates, whereas des[26-84] and des[58-110] are metastable dead-end species that preferentially reshuffle. The key factor in determining the kinetic fate of these des species is the relative accessibility of both their thiol groups and disulfide bonds. Productive intermediates tend to be disulfide-secure, meaning that their structural fluctuations preferentially expose their thiol groups, while keeping their disulfide bonds buried. By contrast, dead-end species tend to be disulfide-insecure, in that their structural fluctuations expose their disulfide bonds in concert with their thiol groups. This distinction leads to four generic types of oxidative folding pathways. We combine these results with those of earlier studies to suggest a general three-stage model of oxidative folding of RNase A and other single-domain proteins with multiple disulfide bonds.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ribonuclease Pancreático
/
Dobramento de Proteína
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
Proc Natl Acad Sci U S A
Ano de publicação:
2001
Tipo de documento:
Article
País de afiliação:
Estados Unidos