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

David S. Libich, Vitali Tugarinov, G. Marius Clore

Research output: Contribution to journalArticlepeer-review

46 Scopus citations

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" GroELbound 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 from15N,1HN,and 13Cmethyl relaxation dispersion experiments, is composed of a prominent, surface-exposed hydrophobic patch.

Original languageEnglish (US)
Pages (from-to)8817-8823
Number of pages7
JournalProceedings of the National Academy of Sciences of the United States of America
Volume112
Issue number29
DOIs
StatePublished - Jul 21 2015

Keywords

  • Chaperonins
  • Dark state exchange saturation transfer
  • Invisible states
  • Lifetime line broadening
  • Relaxation dispersion

ASJC Scopus subject areas

  • General

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