| Project/Area Number |
09279104
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas (A)
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| Allocation Type | Single-year Grants |
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| Research Institution | The University of Tokyo (2000-2001)
Keio University (1997-1999) |
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Principal Investigator |
KAMIMURA Shinji Graduate School of Arts and Sciences, the University of Tokyo Associate Professor, 大学院・総合文化研究科, 助教授 (90177585)
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| Co-Investigator(Kenkyū-buntansha) |
SHIMAMOTO Nobuo National Institute of Genetics Professor, 教授 (20127658)
YANAGIDA Toshio Graduate School of Medicine, Osaka University Professor, 医学系研究科, 教授 (30089883)
KINOSHITA Kazuhiko Institute for Molecular Science, Okazaki National Research Institute Professor, 教授 (30124366)
OIWA Kazuhiro Kansai Advanced Research Center, Communications Research Laboratory Researcher, 通信総合研究所, 室長(研究職)
上田 太朗 工業技術院, 産業技術融合領域研究所, 主任研究員
UYEDA Taro q.p. National Institute for Advanced Interdisciplinary Research Researcher
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| Project Period (FY) |
1997 – 2000
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| Keywords | myosin / dynein / single molecule detection and analysis / biomotor / mechanism of cell motility / chemo-mechanical coupling / F1-ATPase / trp repressor |
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| Research Abstract |
During grant support for the project we executed several different kinds of research works on the biophysical and biochemical features of bio-motors. In particular our efforts were paid to develop novel techniques that enable us to approach directly to single molecular events. K.K. developed a novel technique to detect molecular orientation from fluorescence polarization, which was applied to observe helical motions of actin filaments on myosin fibers. T.Y. improved his technique to detect a single ATP molecule during force development by myosin and the applied experiments showed ca.100 ms of delay for the force generation after ATP binding suggesting an existence of energy-storing state of myosin. He also improved micro glass-needle technique to analyze force-velocity relationship of a single myosin under various experimental conditions. S.K. developed a new technique to record FRET of single molecules with real time resolution (ca.10 Hz time resolution). New trials using various kind
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s of other bio-motors were also carried out during the project. N.S. showed that sliding motion of E. coli trp-repressor on DNA strands enhanced its affinity towards specific DNA sequences and has started single-molecule analysis. Inter-molecular collision among RNA polymerases was also shown by N.S. to be very crucial for the initial formation of short RNA fragments. K.K found that the rotation steps of F1-ATPases being labeled with fluorescent actin was just 120 degrees and the observed speed was as high as 100 Hz. Although the following approaches were not such experiments using single bio-motors but new studies on various types of motor proteins were carried out. S.K. applied a caged-ATP technique to analyze force develapment by axonemal dynein and showed that dynein-ADP was the force-generating intermediate of during ATP hydrolysis. K.O. analyzed detailed features of inner dynein of Chlamydomonas. He found that dynein subspecies f was a motor molecule with an exceptionally high duty-ratio and that subspecies c was a processive motor. T.Q.P.U. found co-operativity between two head domains of myosin from the analysis of single-headed myosin motors. He also found cross bridge could be highly stabilized by G680V mutation in Dictyostelium myosin. Other novel techniques were developed during the project, e.g. novel fluorescent ATP analogues to detect the conformation changes of myosin (K.O.), an artificial system to reconstruct the active directional flow by bio-motors (T.Q.P.U.), a new AFM technique using sensitive-probes to detect surface charge differences (T.Y.), AFM detection of bio-oscillatory motion (S.K.) should be still preliminary but further studies for the applications should be awaited. Less
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