| Project/Area Number |
63470133
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
結晶学
|
|---|
| Research Institution | Osaka University |
|---|
Principal Investigator |
KATSUBE Yukiteru Inst. for Protein Research, Osaka Univ. Prof., たんばく質研究所, 教授 (20032013)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
KITAGAWA Yasuyuki Inst. for Protein Research, Osaka Univ. Assist., たんぱく質研究所, 助手 (70195254)
SATO Mamoru Inst. for Protein Research, Osaka Univ. Assist., たんぱん質研究所, 助手 (60170784)
HATA Yasuo Inst. for Protein Research, Osaka Univ. Assist., たんぱく質研究所, 助手 (10127277)
MATSUURA Yoshiki Inst. for Protein Research, Osaka Univ. Assist. Prof., たんばく質研究所, 助教授 (90029968)
楠木 正巳 大阪大学, たんぱく質研究所, 助手 (90135749)
|
|---|
| Project Period (FY) |
1988 – 1990
|
| Project Status |
Completed (Fiscal Year 1990)
|
| Budget Amount *help |
¥6,000,000 (Direct Cost: ¥6,000,000)
Fiscal Year 1990: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1989: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1988: ¥3,900,000 (Direct Cost: ¥3,900,000)
|
|---|
| Keywords | Protein / Crystal Structure / Structure / Function / ATP Binding Protein / Flexibility / Glutathione Synthetase / イメ-ジングプレ-ト回析計 / X線回析 / 蛋白質結晶解析 / 動的構造解析 / 放射光 / イメ-ジングプレ-ト / 動的講造解析 / イメージングプレート |
|---|
| Research Abstract |
Globular proteins are generally close-packed, but they do have certain degrees of flexibility. Crystallographic studies of changes in structure are restricted by the problem that a structure determined by diffraction methods is averaged over the diffracting volume of the crystal and over the time needed to record the diffraction intensities. Protein crystal data collection by conventional methods can take some months, so that the structural changes occurring on the picosecond to kilosecond time scale escape observation. This can be partially overcome by using a mutant engineered to save the reaction rate and using high speed data-collection techniques.
In a series of studies on flexibility of protein structures, we have developed an Imaging-Plate (IP) diffractometer with a conventional X-ray generator, which is capable of recording diffraction peaks in similar manner to an area-detector and measuring the intensities automatically in succession. The number of reflections which can be measured in an hour by the IP diffractometer is 10 times more than that by a 4-cycles diffractometer.
It has been shown by the crystal-structure determination of glutathione synthetase that the enzyme has a flexible loop, 226-IPQGGETRGNLAAGGGR-242, which undergoes conformational change on binding the substrates. To analyze the role of the flexible loop and a mode of structural change of the loop, we have determined the structures of complexes, enzyme-ATP, enzyme-gamma-Glu-Abu and enzyme-ATP-gamma-Glu-Abu, and the structures of the mutant G240V and the mutant in which the loop was completely deleted, by using the IP diffractometer. Comparison among the structures of the wild-type, the complexes and the mutants has revealed that the flexible loop moves toward the binding sites of the substrates to increase the enzyme specifically, in Particular to exclude water molecule as an acceptor of the phosphoryl group.
|
