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Proc Natl Acad Sci U S A. 2015 Jul 21;112(29):8817-23. doi: 10.1073/pnas.1510083112. Epub 2015 Jun 29.

Intrinsic unfoldase/foldase activity of the chaperonin GroEL directly demonstrated using multinuclear relaxation-based NMR.

Author information

1
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
2
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520 mariusc@mail.nih.gov.

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.

KEYWORDS:

chaperonins; dark state exchange saturation transfer; invisible states; lifetime line broadening; relaxation dispersion

PMID:
26124125
PMCID:
PMC4517251
DOI:
10.1073/pnas.1510083112
[Indexed for MEDLINE]
Free PMC Article

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