Study of dynamic characteristic of muscle proteins by nanosecond time-resolved energy transfer measurements.

这个项目的目标是研究muscle蛋白质的动态特征学，由nanosecond time-resolved能量转移度量。我们修复了纳米秒时间解析的荧光计，其中的荧光计是从名古屋大学物理学系转移过来的。在另一方面，我们准备了突变体肌钙蛋白和肌钙蛋白I，通过表达大肠杆菌的FRET测量。We studed 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イD12+イエD1 concentration。通过这些研究，我们获得了一些重要的数据，以阐明肌氨酸-肌氨酸的调节机制。热带细胞瘤蛋白不会改变其在重新固定的薄膜中的位置，以响应Ca 2+离子集中的变化。结果并不支持关于骨骼肌肉瘦膜的运动的运动，该运动被认为是在静态阻塞理论中。A. T-终端域的C-终端域我移动到抑制期间的外域，而T-终端域的C-终端域的C-终端域并不移动。The CaイイD12+イエ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イD12+イエD1 is very important event during inhibition。Then we have introduced a new model for the Ca-D12 + Ca-D1-mediated regulation of tropomyosin-troponin in skeletal muscle in replace to the steric blocking theory。此外，我们有一些重要的结果来揭示监管机制，我们并不应用生命周期的测量来分析荧光能量转移。因此，我们需要继续这个项目来开发一种流动能量转移的方法。

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.