Intrinsic unfoldase/foldase activity of the chaperonin GroEL directly demonstrated using multinuclear relaxation-based NMR.
Proc Natl Acad Sci U S A
; 112(29): 8817-23, 2015 Jul 21.
Article
em En
| MEDLINE
| ID: mdl-26124125
ABSTRACT
The prototypical chaperonin GroEL assists protein folding through an ATP-dependent encapsulation mechanism. The details of how GroEL folds proteins remain elusive, particularly because encapsulation is not an absolute requirement for successful re/folding. Here we make use of a metastable model protein substrate, comprising a triple mutant of Fyn SH3, to directly demonstrate, by simultaneous analysis of three complementary NMR-based relaxation experiments (lifetime line broadening, dark state exchange saturation transfer, and Carr-Purcell-Meinboom-Gill relaxation dispersion), that apo GroEL accelerates the overall interconversion rate between the native state and a well-defined folding intermediate by about 20-fold, under conditions where the "invisible" GroEL-bound states have occupancies below 1%. This is largely achieved through a 500-fold acceleration in the folded-to-intermediate transition of the protein substrate. Catalysis is modulated by a kinetic deuterium isotope effect that reduces the overall interconversion rate between the GroEL-bound species by about 3-fold, indicative of a significant hydrophobic contribution. The location of the GroEL binding site on the folding intermediate, mapped from (15)N, (1)HN, and (13)Cmethyl relaxation dispersion experiments, is composed of a prominent, surface-exposed hydrophobic patch.
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Texto completo:
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Dobramento de Proteína
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Chaperonina 60
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Ressonância Magnética Nuclear Biomolecular
Limite:
Animals
Idioma:
En
Revista:
Proc Natl Acad Sci U S A
Ano de publicação:
2015
Tipo de documento:
Article