Equilibrium and kinetics of the allosteric transition of GroEL studied by solution X-ray scattering and fluorescence spectroscopy

Tomonao Inobe, Munehito Arai, Masaharu Nakao, Kazuki Ito, Kiyoto Kamagata, Tadashi Makio, Yoshiyuki Amemiya, Hiroshi Kihara, Kunihiro Kuwajima

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

We have studied the ATP-induced allosteric structural transition of GroEL using small angle X-ray scattering and fluorescence spectroscopy in combination with a stopped-flow technique. With X-ray scattering one can clearly distinguish the three allosteric states of GroEL, and the kinetics of the transition of GroEL induced by 85μM ATP have been observed directly by stopped-flow X-ray scattering for the first time. The rate constant has been found to be 3-5s-1 at 5°C, indicating that this process corresponds to the second phase of the ATP-induced kinetics of tryptophan-inserted GroEL measured by stopped-flow fluorescence. Based on the ATP concentration dependence of the fluorescence kinetics, we conclude that the first phase represents bimolecular non-cooperative binding of ATP to GroEL with a bimolecular rate constant of 5.8×105M-1s-1 at 25°C. Considering the electrostatic repulsion between negatively charged GroEL (-18 of the net charge per monomer at pH 7.5) and ATP, the rate constant is consistent with a diffusion-controlled bimolecular process. The ATP-induced fluorescence kinetics (the first and second phases) at various ATP concentrations (<400μM) occur before ATP hydrolysis by GroEL takes place and are well explained by a kinetic allosteric model, which is a combination of the conventional transition state theory and the Monod-Wyman-Changeux model, and we have successfully evaluated the equilibrium and kinetic parameters of the allosteric transition, including the binding constant of ATP in the transition state of GroEL.

Original languageEnglish
Pages (from-to)183-191
Number of pages9
JournalJournal of Molecular Biology
Volume327
Issue number1
DOIs
Publication statusPublished - 2003 Mar 14
Externally publishedYes

Keywords

  • Allosteric effect
  • Chaperonin
  • Cooperativity
  • Stopped-flow
  • X-ray scattering

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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