2007 Fiscal Year Final Research Report Summary
Kinetic Studie on the Functional Expression of Chaperonin
| Project/Area Number |
17370052
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | National Institutes of Natural Sciences Okazaki Research Facilities (2007)
The University of Tokyo (2005-2006) |
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Principal Investigator |
KUWAJIMA Kunihiro National Institutes of Natural Sciences Okazaki Research Facilities, National Institutes of Natural Sceince, Okazaki Institute for Integrative Bioscience, Professor (70091444)
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| Co-Investigator(Kenkyū-buntansha) |
MAKI Kousuke Nagoya University, Graduate School of Science, Associate Professor (30361570)
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| Project Period (FY) |
2005 – 2007
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| Keywords | molecular chaperone / chaperonin / solution X-ray scattering / allosteric transition / folding / ATP binding |
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| Research Abstract |
The objective of the present study was to elucidate quantitatively and physicochemically the molecular mechanism of chaperonin functions by means of physical measurements like small-angle X-ray scattering and kinetic measurements like a stopped-flow technique. The following results were obtained.
Although the chaperonin GroEL and GroES from Escherichia coli form the GroEL/GroES complex, its stoichiometry, namely, whether they form a 1: 1 molar-ratio bullet-type complex or rather a 1: 2 molar-ratio football-type complex, was not clear. We used the solution X-ray scattering technique to directly observe the structure of the GroEL/GroES complex in solution under a physiological condition. The results have shown that the bullet-type complex is predominant and that the football-type complex is not present in a significant amount.
The multi-sigmoidal behavior of the ATP-concentration dependence of the rate constant of the ATP-induced allosteric transition of the chaperonin GroEL has been interpreted in terms of the double-ring structure of GroEL. In the present study, however, we found that a single-ring mutant(SR1) of GroEL exhibited similar multi-sigmoidal behavior of its ATP dependence. This strongly suggests the presence of at least two binding sites for ATP on GroEL. ADP inhibited the ATP-induced allosteric transition of GroEL, and from the dependence of the apparent rate constant of the allosteric transition on ADP, the second ATP-binding site on GroEL was occupied by ADP at 100 μM ADP and 400 μM of ATP. Under the condition but using azido-ADP instead of ADP, we photochemically labeled GroEL. We confirmed the presence of a phosphate group in the photoaffinity labeled GroEL thus obtained using a fluorescent zinc complex, demonstrating the presence of the second ATP-binding site. We are going to identify the location of the second binding site along the amino acid sequence of GroEL.
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