| Project/Area Number |
09044217
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Field |
Bioproduction chemistry/Bioorganic chemistry
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
ODA Jun'ichi KYOTO UNIVERSITY Inst.Chem.Res., Professor, 化学研究所, 教授 (50027041)
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| Co-Investigator(Kenkyū-buntansha) |
NAKATSU Toru KYOTO UNIVERSITY Inst.Chem.Res., Associate Instructor, 化学研究所, 教務職員 (50293949)
KATO Hiroaki KYOTO UNIVERSITY Inst.Chem.Res., Instructor, 化学研究所, 助手 (90204487)
WAKATSUKI Soichi ESRF,Beamline Corespondence, ビームライン責任者
SCHLICHTING Ilme Max-Planck_institute for Mol.Physiol., Group Reader, グループ主任
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| Project Period (FY) |
1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1997: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Keywords | time-resolved crystallography / enzyme / protein crystallography / caged compound / synchronization of reaction / Laue diffraction / 時分割X線結晶解析 / タンパク質結晶学 |
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| Research Abstract |
To investigate the action of functional proteins such as enzymes, it is essential to visualize their structure in motion directly. In this studies, we aim to capture the short-lived productive Michaelis complex structure during the reaction of glutathione synthetase. To accomplish this, we used a transition-state analogue inhibitor (TSA) which is subjected to enzyme-catalyzed phosphorylation by ATP within the enzyme active site as a substrate of enzyme. We thus crystallized the enzyme complexed with this inhibitor and a caged-ATP to prevent the phosphorylation. This system allows to release ATP upon photdlysis to initiate the phosphorylation of the inhibitor within the enzyme active site. We used white beam Laue diffraction method or conventional monochromatic methods with flash cooling. We planed to use the monochromatic methods to compensate the results of Laue methods since the Laue diffraction has two disadvantages, low completeness of the low resolution data and high X-ray damage with crystals. We succeed to measure very good Laue diffraction data at the beamline ID9 at European Synchrotron Radiation Facility (ESRF). We also archived to install the computer program developed at ESRF to handle the Laue diffraction in our laboratory at Institute for Chemical Research, Kyoto University. The results showed that we have captured the structure of TSA unphosphorylated at immediately after photolysis. We also determine time-resolved movies during the TSA is being phosphorylated with ATP at the enzyme active site.
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