title: Intrinsic unfoldase/foldase activity of the chaperonin GroEL directly demonstrated using multinuclear relaxation-based NMR.
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.
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: David S
authors.initials: DS
authors.lastname: Libich
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
authors.forename: Vitali
authors.initials: V
authors.lastname: Tugarinov
authors.affiliation: 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.
authors.forename: G Marius
authors.initials: GM
authors.lastname: Clore
mesh_terms: D000818:Animals; D002247:Carbon Isotopes; D018834:Chaperonin 60 / Q000737:chemistry* / Q000378:metabolism*; D002645:Chickens; D007700:Kinetics; D008958:Models, Molecular; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D018909:src Homology Domains
chemical_list: D002247:Carbon Isotopes; D018834:Chaperonin 60; D009587:Nitrogen Isotopes
keywords: chaperonins; dark state exchange saturation transfer; invisible states; lifetime line broadening; relaxation dispersion