2001 Fiscal Year Final Research Report Summary
Structural Analysis of Dymamics of Celluar Motors
| Project/Area Number |
09279103
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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 |
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Principal Investigator |
SUTOH Kazuo Graduate School of Arts and Sciences, The University of Tokyo, Professor, 大学院・総合文化研究科, 教授 (20111453)
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| Co-Investigator(Kenkyū-buntansha) |
WAKABAYASHI Takeyuki Graduate School of Science, The University of Tokyo, Professor, 大学院・理学系研究科, 教授 (90011717)
WAKABAYASHI Katsuzo Osaka University, Graduate School of Engineering Science, Professor, 大学院・基礎工学研究科, 教授 (00029521)
SHIRAKIHARA Yasuo National Institute of Genetics, Associate Professor, 構造遺伝学研究センター, 助教授 (20150287)
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| Project Period (FY) |
1997 – 2000
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| Keywords | Myosin / Dynein / Kinesin / FRET / X-ray scattering / Electron microscopy / Recombinant motors / X-ray crystallography |
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| Research Abstract |
Based on crystal structures of the motor domain of Dictyostelium myosin II, we carried out of the structure-function analysis of this motor domain. We constructed many types of Dictyostelium myosin II mutants, which were trapped at a specific step of ATP hydrolysis cycle. Biochemical and biophysical studies on these mutants have shown how the three-dimensional structure of the motor domain rearranges at each step of ATP hydrolysis cycle, and how these changes are finally transmitted to the actin-binding site and the converter domain to generate sliding and force generation on an actin filament. Furthermore, we carried out the GFP-based FRET measurements of the fusion protein consisting of GFP, the motor domain and BFP. These FRET measurements have shown that the converter domain really swings at the isomerization step first and then swings back at the Pi release step. The latter step is likely to correspond to the stroke step. We have then constructed a prism-based total reflection fluorescence microscope that can visualize fluorescence spectra of many single molecules in a microscope filed at the same time at the video rate. By using this novel technique, we have detected that the converter of myosin motor domain is trapped in a particular orientation in the presence of various types of nucleotide analogs such as MgADP.Vi, MgADP.BeFx and MgADP.AlFx, consistent with the ensemble measurements mentioned above.
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