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Unusual Structure and Function of Active Intermediate of Myosin Realized during Sliding Movement : A Possibility for Energy Storage State

滑动过程中肌球蛋白活性中间体的异常结构和功能：能量储存状态的可能性

基本信息

批准号：
16370070
负责人：
KATAYAMA Eisaku
金额：
$ 8.7万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

As a means to investigate the 3-D structural changes of sliding actomyosin, we have developed a "differential pattern-recognition method" to quantitatively compare the surface profiles of the protein assembly instantaneously captured. by quick-freeze deep-etch replication, with those of the artificial images simulated from the atomic models of the target. We discovered that actin-attached myosin head during sliding takes a configuration kinked to the opposite direction from widely believed ATP-bound form. Since we also found the presence of particles in the same configuration but with different attachment angle to actin, we confirmed the significance of that novel structure, and proposed a new scheme of crossbridge movement, that is quite different from the conventional one, but comprehensively accounts for the presence of all the observed structures. We also analyzed the structures of actin-attached myosin-V motor-domain with long lever-arm moiety, during processive walking movement a … More long actin-filament, by a similar means. The trail head showed a rigor-like pattern as postulated in a preceding study, but the images of the lead heads matched by far better with our novel structure than ATP-bound form postulated in the above study, suggesting that the structural change we found in myosin-II might be universally applicable to unconventional myosin species. We are also developing a novel multifunctional microscopic probe to indicate the localization of specific sites or subdomains in the protein assembly, which might enable us to follow the dynamic behavior of given molecules even within intracellular environments. We successfully fused the probe construct to myosin head and attempted to visualize the movement of the probe using the world best-rated atomic force microscope with highest temporal and spatial resolutions. However, the movement was too fast and subtle and the material was not well-immobilized onto mica surface. We would retry after enhancing the adsorption and introducing multiple probes in a molecule. Less

作为一种手段来调查的3-D结构变化的滑动肌动球蛋白，我们已经开发了一种“差分模式识别方法”，以定量比较瞬时捕获的蛋白质组装的表面轮廓。通过快速冷冻深蚀刻复制，与那些从目标的原子模型模拟的人工图像。我们发现，肌动蛋白附着的肌球蛋白头在滑动过程中采取的配置扭结到相反的方向，从广泛认为的ATP结合的形式。由于我们还发现了具有相同构型但与肌动蛋白具有不同附着角的粒子的存在，我们证实了这种新结构的意义，并提出了一种新的过桥运动方案，该方案与传统方案截然不同，但全面解释了所有观察到的结构的存在。我们还分析了肌动蛋白附着的肌球蛋白-V运动域的结构，在进行性步行运动和 ...更多信息长肌动蛋白丝，通过类似的方法。如先前研究中所假设的，尾状头显示出类似于僵硬的模式，但铅头的图像与我们的新结构的匹配程度远远高于上述研究中假设的ATP结合形式，这表明我们在肌球蛋白II中发现的结构变化可能普遍适用于非常规肌球蛋白物种。我们还正在开发一种新的多功能显微探针，以指示蛋白质组装中特定位点或子域的定位，这可能使我们能够跟踪给定分子的动态行为，即使在细胞内环境中。我们成功地将探针结构融合到肌球蛋白头上，并尝试使用世界上最好的原子力显微镜以最高的时间和空间分辨率可视化探针的运动。然而，该运动太快且太细微，并且该材料没有很好地固定在云母表面上。我们将在增强吸附并在分子中引入多个探针后重试。少