| Project/Area Number |
04454619
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物物性学
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
MIKI Kunio Tokyo Inst.of Tech., Res.Lab.of Resources Utilization, Associate Professor, 資源化学研究所, 助教授 (10116105)
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| Co-Investigator(Kenkyū-buntansha) |
INAKA Koji Tokyo Inst.of Tech., Res.Lab.of Resources Utilization, Research Associate, 資源化学研究所, 助手 (30240758)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1993: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1992: ¥5,000,000 (Direct Cost: ¥5,000,000)
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| Keywords | Chaperonin / Heat-shock protein / Protein refolding / Crystallization / X-ray crystal structure analysis / Synchrotron radiation / Thermophilic bacteria |
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| Research Abstract |
We have purified and crystallized chaperonin (hsp60), a molecular chaperon protein, from a thermophilic bacterium, Thermus thermophilus, in order to determine its three-dimensional structure by X-ray crystallography.
We obtained two forms of chaperonin crystals, plates and hexagonal prisms by the use of polyethyleneglycol as a precipitant. The diffraction studies on the macromolecular Weissenberg camera by synchrotron radiation at Photon Factory showed that the former and latter crystals diffracted X-rays up to 10 and 7 A resolution, respectively. The plate and hexagonal prism crystals belong to the monoclinic space group P2 or P2_1 and hexagonal space group P6_322, respectively. The unit-cell parameters of both crystals were also determined.
The SDS and native polyacrylamide gel electrophoreses of these crystals revealed that the plate crystals contain the holo-chaperonin complex composed of cpn60 14mer and cpn10 7mer. On the other hand, it was found that hexagonal crystals are composed of the monomeric 50KDa fragments which were thought to be occurred by natural proteolysis of cpn60.
As resolution limit for X-ray diffraction of the 50kDa fragment was not so high, the crystallization was performed for the following two modified samples ; 1) the purified 50KDa fragment and 2) the 50KDa fragment treated by trypsin. Crystals with different unit-cell dimensions were obtained but their resolution limit does not reach atomic resolution.
The further improvement of resolution limits of these crystals is still underway.
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