| Project/Area Number |
10680633
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Biophysics
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| Research Institution | Osaka University |
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Principal Investigator |
ARATA Toshiaki Graduate School of Science, Osaka Univ. Research Associate, 大学院・理学研究科, 助教授 (70151165)
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| Co-Investigator(Kenkyū-buntansha) |
KATSUZOU Wakabayashi Graduate School of Engineering Science, Osaka Univ.Professor, 大学院・基礎工学研究科, 教授 (00029521)
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2000: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1999: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1998: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | monomeric actin / myosin head / kinesin / tubulin / small-angle x-ray scattering / electron spin resonance / crystallography / spin label / 非重合アクチン / アクトミオシン |
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| Research Abstract |
We made a nonpolymerizable G-actin by MBS and DHT and measured X-ray solution scattering from a 1 : 1 acto-myosin S1 complex. The radius of gyration (Rg) of the complex was 49 A and apparent molecular weight was 165 kD.The model consisted of atomic structures of actin and S1 which fits the whole scattering curve closely was searched on the basis of rigid-body rotation and translation of two molecules. The uniqueness of the model was checked by a complex of an asymmetric DNaseI-actin and S1. A tip of S1 binds to actin subdomain 1 as expected from the previous electron microscopy of a myosin molecule whose heads are decorated with a monomeric G-actin (J.Struct.Biol. ('98)123, 8). In the presence of ADP, Rg decreased by 3 A with no change in molecular weight. The best fit model was obtained by twisting the two molecules each other through nearly 90 degrees about a long axis of S1 with retaining their main binding site. The complex of dimeric or timeric actin and S1 is now examined, and crystallization is in progress. The computer-fitting of ESR spectra from spin-labeled myosin light-chain in muscle fibres was done by a combination of two equimolar Gaussian distributions. The spectrum of relaxed muscle fibres was fitted with an asymmetric distribution and resolved into two broad angular distributions, suggesting that a pair of the myosin light-chain domains (neck regions) appear fixed loosely at different orientations on the myosin filament backbone. The spectrum from active muscle fibres is now analyzed. The tubulin-binding region of kinesin was spin-labeled using site-directed mutagenesis. A side-chain mobility estimated from spin-label ESR was restricted by tubulin binding, The immobilization depends on the occupancy of kinesin on a microtubule, suggesting a propagation of structural change along microtubule.
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