2000 Fiscal Year Final Research Report Summary
Study of functions of motor protein by protein engineering and holographic electron cryo-microscopy
| Project/Area Number |
09102006
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| Research Category |
Grant-in-Aid for Specially Promoted Research
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| Allocation Type | Single-year Grants |
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| Review Section |
Biological Sciences
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| Research Institution | University of Tokyo |
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Principal Investigator |
WAKABAYASHI Takeyuki University of Tokyo, School of Science, Professor, 大学院・理学系研究科, 教授 (90011717)
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| Co-Investigator(Kenkyū-buntansha) |
YASUNAGA Takuo University of Tokyo, School of Science, Research Associate, 大学院・理学系研究科, 助手 (60251394)
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| Project Period (FY) |
1997 – 2000
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| Keywords | Muscle confraction / protein engineering / electron cryo-microscopy / motor protein / actin / myosin / Ca regulation / X-ray crystallography |
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| Research Abstract |
The electron cryo-microscopy is the most promising method to visualize proteins under the physiological conditions. Because the amplitude contrast produced by the frozen hydrated proteins is low, phase-contrast should be increased with large defocus. This requires that the spatial coherence of electron beam is good. We could compensate the blurring due to underfocus using the holographic image reconstruction technique(HIRT) we developed.
We applied this method to visualize the three-dimensional structure of thin filaments and showed the calcium-induced changes of troponin. We reconstructed three-dimensional structure of actin-tropomyosin-troponin complex from rabbit skeletal muscle by electron cryo microscopy and image analysis using back projection. We found the mass of troponin head over the inner domain of actin in the presence of Ca^<2+>. On the other hand, troponin covered the frontal surface of actin in the absence of Ca^<2+> including the C-terminal region by troponin-arm. Also t
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ropomyosin was found to be shifted differentially depending upon Ca ions. We proposed a new model of calcium regulation of muscle contraction from these new data.
We use protein engineering to produce the mutant actins that activate myosin ATPase in a presence of tropomyosin-troponin and calcium much higher than the wild-type actin. We found that the replacement of single amino acid alanine230 to tyrosine is sufficient to produce this effect. We solved the atomic structure of the wild-type actin and mutant ones and found that the side chain of leucine236 is more exposed to solvent. The water structure in the Ca^<2+> ATP binding site suggests that actin recognizes the hydrated form of the adenine ring of ATP.
We developed a move method to determine the three-dimensional positions of fluorophores by combining the FRET data and other structural information available. Using this method, we could determine the ATP-induced changes of three-dimensional structure of truncated Dictyostelium myosin in solution. Also the method elucidated actin Cys374 relocation induced by labelling of fluorescent dyes. Less
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