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，我们发展了一种“差异模式识别方法”来定量比较瞬时捕获的蛋白质组装的表面轮廓。通过快速冷冻深刻蚀复制，用人造图像模拟靶材的原子模型。我们发现，在滑动过程中，肌动蛋白附着的肌球蛋白头部的构型与人们普遍认为的ATP结合形式相反。由于我们还发现了相同构型但不同附着角度的颗粒对肌动蛋白的存在，我们确认了这种新结构的意义，并提出了一种新的跨桥运动方案，它与传统的方案有很大的不同，但综合考虑了所有观察到的结构的存在。我们还分析了肌动蛋白连接的肌球蛋白-V运动域的长臂部分，在前进的步行运动中的一个…的结构更长的肌动蛋白细丝，用类似的方法。轨迹头部呈现出先前研究中假设的僵直样图案，但铅头部的图像与我们的新结构比上述研究中假设的ATP结合形式更符合我们的新结构，这表明我们在Myosin-II中发现的结构变化可能普遍适用于非传统肌球蛋白物种。我们还在开发一种新型的多功能显微探针，以指示蛋白质组装中特定位置或亚域的定位，这可能使我们能够跟踪给定分子的动态行为，甚至在细胞内环境中也是如此。我们成功地将探针结构融合到肌球蛋白头部，并尝试使用具有最高时间和空间分辨率的世界最佳原子力显微镜来可视化探针的运动。然而，移动太快和太微妙，材料没有很好地固定在云母表面。我们将在增强吸附并在一个分子中引入多个探针后重试。较少

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