title: The H/D-exchange kinetics of the Escherichia coli co-chaperonin GroES studied by 2D NMR and DMSO-quenched exchange methods.
abstract: We studied hydrogen/deuterium-exchange reactions of peptide amide protons of GroES using two different techniques: (1) two-dimensional (1)H-(15)N transverse-optimized NMR spectroscopy and (2) the dimethylsulfoxide-quenched hydrogen-exchange method combined with conventional (1)H-(15)N heteronuclear single quantum coherence spectroscopy. By using these techniques together with direct heteronuclear single quantum coherence experiments, we quantitatively evaluated the exchange rates for 33 out of the 94 peptide amide protons of GroES and their protection factors, and for the remaining 61 residues, we obtained the lower limits of the exchange rates. The protection factors of the most highly protected amide protons were on the order of 10(6)-10(7), and the values were comparable in magnitude to those observed in typical small globular proteins, but the number of the highly protected amide protons with a protection factor larger than 10(6) was only 10, significantly smaller than the numbers reported for the small globular proteins, indicating that significant portions of free heptameric GroES are flexible and natively unfolded. The highly protected amino acid residues with a protection factor larger than 10(5) were mainly located in three β-strands that form the hydrophobic core of GroES, while the residues in a mobile loop (residues 17-34) were not highly protected. The protection factors of the most highly protected amide protons were orders of magnitude larger than the value expected from the equilibrium unfolding parameters previously reported, strongly suggesting that the equilibrium unfolding of GroES is more complicated than a simple two-state or three-state mechanism and may involve more than a single intermediate.
authors.affiliation: Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.
authors.forename: Mahesh S
authors.initials: MS
authors.lastname: Chandak
authors.forename: Takashi
authors.initials: T
authors.lastname: Nakamura
authors.forename: Koki
authors.initials: K
authors.lastname: Makabe
authors.forename: Toshio
authors.initials: T
authors.lastname: Takenaka
authors.forename: Atsushi
authors.initials: A
authors.lastname: Mukaiyama
authors.forename: Tapan K
authors.initials: TK
authors.lastname: Chaudhuri
authors.forename: Koichi
authors.initials: K
authors.lastname: Kato
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Kuwajima
mesh_terms: D018835:Chaperonin 10 / Q000737:chemistry* / Q000378:metabolism*; D041961:Deuterium Exchange Measurement; D004121:Dimethyl Sulfoxide / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D058767:Protein Unfolding
chemical_list: D018835:Chaperonin 10; D029968:Escherichia coli Proteins; D006859:Hydrogen; D004121:Dimethyl Sulfoxide