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

作为研究滑动肌动球蛋白三维结构变化的一种手段，我们开发了一种“差分模式识别方法”来定量比较即时捕获的蛋白质组装的表面轮廓。通过速冻深蚀刻复制，用这些人造图像模拟出目标的原子模型。我们发现，在滑动过程中，肌动蛋白附着的肌凝蛋白头部的结构与普遍认为的atp结合形式相反。由于我们还发现了相同构型但与肌动蛋白的附着角度不同的粒子的存在，我们证实了这种新结构的意义，并提出了一种新的跨桥运动方案，该方案与传统的方案有很大不同，但全面考虑了所有观察到的结构的存在。我们还用类似的方法分析了肌动蛋白附着的具有长杠杆臂部分的肌球蛋白- v运动结构域在进步性行走运动中的结构，以及更长的肌动蛋白丝。与之前的研究一样，尾头显示出类似严谨的模式，但与上述研究中假设的atp结合形式相比，头头的图像与我们的新结构匹配得更好，这表明我们在肌凝蛋白ii中发现的结构变化可能普遍适用于非常规肌凝蛋白物种。我们还在开发一种新的多功能显微探针，用于指示蛋白质组装中特定位点或亚结构域的定位，这可能使我们能够在细胞内环境中跟踪给定分子的动态行为。我们成功地将探针结构融合到肌球蛋白头部，并尝试使用世界上最好的原子力显微镜观察探针的运动，具有最高的时间和空间分辨率。然而，移动速度太快，动作过于细微，材料在云母表面的固定效果不佳。我们会在增强吸附和在一个分子中引入多个探针后重试。少

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