| Project/Area Number |
15370069
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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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 | School of Science and Engineering, Teikyo University |
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Principal Investigator |
WAKABAYASHI Takeyuki School of Science Teikyo University, Department of Biosciences, Professor, 理工学部, 教授 (90011717)
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| Project Period (FY) |
2003 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥15,100,000 (Direct Cost: ¥15,100,000)
Fiscal Year 2005: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2004: ¥4,100,000 (Direct Cost: ¥4,100,000)
Fiscal Year 2003: ¥7,000,000 (Direct Cost: ¥7,000,000)
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| Keywords | Electron Cryo-Microscopy / NMR spectroscopy / Muscle Contraction / Calcium / Troponin / Actin / Tropomyosin / Cardiomyopathy / カルシウム制御 / 分子モーター / カルシウム・スイッチ / ミオシン / NMR分光 / 電子顕微鏡 |
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| Research Abstract |
Troponin and tropomyosin on actin filaments constitute a Ca^<2+>-sensitive switch that regulates the contraction of vertebrate striated muscle through a series of conformational changes within actin-based thin filament. Troponin consists of three subunits : an inhibitory subunit (TnI), a Ca^<2+>-binding subunit (TnC), and a tropomyosin-binding subunit (TnT). Ca^<2+>-binding to TnC is believed to weaken interactions between troponin and actin, and triggers a large conformational change of the troponin complex. However, the atomic details of the actin-binding sites of troponin have not been determined. Ternary troponin complexes have been reconstituted from recombinant chicken skeletal TnI, TnC, and TnT_2 (the C-terminal region of TnT), among which only TnI was uniformly labelled with ^<15>N and/or ^<13>C. By applying NMR spectroscopy, the solution structures of a ‘mobile' actin-binding domain (〜6.1 kDa) in the troponin ternary complex (〜52 kDa) were determined. The mobile domain appears
… More
to tumble independently of the core domain of troponin. Ca^<2+>-induced changes in the chemical shift and line shape suggested that its tumbling was more restricted at high Ca^<2+>. The atomic details of interactions between actin and the mobile domain of troponin were defined by docking the mobile domain into the cryo-EM density map of thin filament at low Ca^<2+>. This allowed the determination of the 3D position of residue 133 of TnI, which has been an important landmark to incorporate the available information. This enabled unique docking of the entire globular head region of troponin into the thin filament cryo-EM map at low Ca^<2+>. The resultant atomic model suggests that troponin electrostatically interacted with actin and caused the shift of tropomyosin to achieve muscle relaxation. An important feature is that the coiled-coil region of troponin pushed tropomyosin at low Ca^<2+>. Moreover, the relationship between myosin and the mobile domain on actin filaments suggests that the latter works as a fail-safe latch. Less
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