Study of dynamic characteristic of muscle proteins by nanosecond time-resolved energy transfer measurements.
通过纳秒时间分辨能量转移测量研究肌肉蛋白的动态特性。
基本信息
- 批准号:10680629
- 负责人:
- 金额:$ 0.32万
- 依托单位:
- 依托单位国家:日本
- 项目类别:Grant-in-Aid for Scientific Research (C)
- 财政年份:1998
- 资助国家:日本
- 起止时间:1998 至 1999
- 项目状态:已结题
- 来源:
- 关键词:
项目摘要
The purpose of this project is studying of dynamic characteristics of muscle proteins by nanosecond time-resolved energy transfer measurements. We repaired the nanosecond time-resolved fluorometer which was transferred from Department of Physics, Nagoya University. On the other hand, we prepared mutants tropomyosin and troponin I by expressing in Escherichia coli for FRET measurements. We studied the spatial relationships between fluorescent probes attached to specific residues on actin, tropomyosin and troponin and then measured the distance changes between fluorescent probes in response to a change in CaィイD12+ィエD1 concentration. Through these studies, we obtained several important data for understanding the regulation mechanism by troponin-tropomyosin. Tropomyosin does not change its position on the reconstituted thin filament in response to a change in Ca2+ ion concentration. The results do not support the notion of tropomyosin movement on skeletal muscle thin filaments as proposed in the steric blocking theory. The C-terminal domain of troponin I moves to the outer domain of actin during inhibition, while the C-terminal domain of troponin C does not move much. The CaィイD12+ィエD1-induced changes in tropomyosin-troponin complex seems to occur only when F-actin is present, suggesting that a stable complex formation of troponin I with the outer domain of F-actin upon removal of CaィイD12+ィエD1 is very important event during inhibition. Then we have introduced a new model for the CaィイD12+ィエD1-mediated regulation of tropomyosin-troponin in skeletal muscle in replace to the steric blocking theory. Although we got several important results for revealing the regulation mechanism, we do not apply the lifetime measurements to analyze the fluorescence energy transfer. Therefore, we have to continue this project to develop the methods of fluorescence energy transfer.
本项目的目的是通过纳秒时间分辨能量传递测量来研究肌肉蛋白质的动态特性。我们修理了从名古屋大学物理系搬来的纳秒时间分辨荧光仪。另一方面,我们通过在大肠杆菌中表达的方法制备了突变体原肌球蛋白和肌钙蛋白I,用于FRET检测。我们研究了附着在肌动蛋白、原肌球蛋白和肌钙蛋白上特定残基上的荧光探针之间的空间关系,然后测量了荧光探针之间的距离随着CaィイD12+ィエD1浓度的变化而变化。通过这些研究,我们获得了一些了解肌钙蛋白原肌球蛋白调控机制的重要数据。原肌球蛋白不会因钙离子浓度的变化而改变其在重组细丝上的位置。这些结果并不支持空间位阻理论中提出的原肌球蛋白在骨骼肌细丝上运动的观点。肌钙蛋白I的C末端结构域在抑制过程中移动到肌动蛋白的外部结构域,而肌钙蛋白C的C末端结构域移动不多。钙ィイD12+ィエD1诱导的原肌球蛋白-肌钙蛋白复合体的变化似乎只有在F-肌动蛋白存在的情况下才会发生,这表明当去除CaィイD12+ィエD1时,肌钙蛋白I与F-肌动蛋白的外区形成稳定的复合体是抑制过程中非常重要的事件。然后,我们引入了一种新的模型来替代空间位阻理论,即CaィイD12+ィエD1介导的骨骼肌原肌球蛋白-肌钙蛋白的调节。虽然我们已经得到了一些重要的结果来揭示荧光的调节机制,但我们并没有用寿命测量来分析荧光能量转移。因此,我们必须继续这个项目来发展荧光能量转移的方法。
项目成果
期刊论文数量(12)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Miki,M.: "Structural changes between regulatory proteins and actin: A regulation model by tropomyosin-troponin based on FRET measurements in Molecular Interactions of Actin (dos Remedios, C.G. and Thomas, D.D., eds)"Springer Verlag,Heidelberg (in press).
Miki,M.:“调节蛋白和肌动蛋白之间的结构变化:基于肌动蛋白分子相互作用中 FRET 测量的原肌球蛋白-肌钙蛋白调节模型(dos Remedios,C.G. 和 Thomas,D.D.,编辑)”Springer Verlag,海德堡(正在出版)
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Miki,M.et al.: "Ca^<2+>-induced distance change between points on actin and troponin in skeletal muscle thin filaments estimated by fluorescence energy transfer spectroscopy."J.Biohem. 123. 324-331 (1998)
Miki,M.等人:“通过荧光能量转移光谱估计骨骼肌细丝中肌动蛋白和肌钙蛋白上的点之间的Ca ^ 2 诱导的距离变化。”J.Biohem。
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Miki, M., Kobayashi, T., Kimura, H., Hagiwara, A., Hai, H., and Maeda, Y.: "CaィイD12+ィエD1-induced distance change between points on actin and troponin in skeletal muscle thin filaments estimated by fluorescence energy transfer spectroscopy"J. Biochem.. 123
Miki, M.、Kobayashi, T.、Kimura, H.、Hagiwara, A.、Hai, H. 和 Maeda, Y.:“CaliD12+IeD1 引起的骨骼肌细丝中肌动蛋白和肌钙蛋白点之间的距离变化荧光能量转移光谱法估计”J. Biochem.. 123
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Miki, M. et al.: "Fluorescence resonance energy transfer between points on tropomyosin and actin in skeletal muscle thin filaments : Does tropomyosin move?"J. Biochem. 123. 1104-1111 (1998)
Miki, M. 等人:“骨骼肌细丝中原肌球蛋白和肌动蛋白上的点之间的荧光共振能量转移:原肌球蛋白会移动吗?”J.
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- 影响因子:0
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Miki,M.et al.: "Fluorescence resonance energy transfer between points on tropomyosin and actin in skeletal muscle thin filaments: Does tropomyosin move?."J.Biohem. 123. 1104-1111 (1998)
Miki,M.等人:“骨骼肌细丝中原肌球蛋白和肌动蛋白上的点之间的荧光共振能量转移:原肌球蛋白会移动吗?”J.Biohem。
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MIKI Masao其他文献
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{{ truncateString('MIKI Masao', 18)}}的其他基金
Construction of an atomic model of the skeletal muscle thin filament by FRET measurements
通过 FRET 测量构建骨骼肌细丝原子模型
- 批准号:
21570162 - 财政年份:2009
- 资助金额:
$ 0.32万 - 项目类别:
Grant-in-Aid for Scientific Research (C)
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