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

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


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • Animals
  • Carbon Isotopes
  • Chaperonin 60 / chemistry*
  • Chaperonin 60 / metabolism*
  • Chickens
  • Kinetics
  • Models, Molecular
  • Nitrogen Isotopes
  • Nuclear Magnetic Resonance, Biomolecular*
  • Protein Binding
  • Protein Conformation
  • Protein Folding*
  • src Homology Domains


  • Carbon Isotopes
  • Chaperonin 60
  • Nitrogen Isotopes