Cooperativity in Actin Filament and Its Functional Implication : Structural analyses by Quick-Freeze Deep-Etch Replica Electron Microscopy

本研究旨在用快速冷冻深蚀刻电子显微镜捕捉滑动肌球蛋白的瞬时结构，并分析被认为是肌球蛋白运动的简单轨迹的交叉桥和肌动蛋白细丝的三维结构变化。在完成了复制标本的三维重建的创新方法后，我们开始将类似的过程应用于像阴性染色的标本这样的块状固体结构的重建。我们还设计了另一种方法，利用蛋白质分子的原子坐标来预测预期的深蚀刻复制品图像。僵直复合体中肌球蛋白头部的结构与通过对接肌动蛋白和肌球蛋白S1的晶体结构而提出的模型非常匹配。然而，与预测不同的是，大多数滑动条件下的交叉桥的构型与ATP结合的交叉桥的构型扭转方向相反。由于观察到的…更多的结构出现了与PPDM-交联型肌球蛋白/ADP复合物非常相似的结构，我们利用新的模式匹配方法对复制图像和模拟图像的表面轮廓进行了定量比较。结果清楚地证实了肌球蛋白头在滑动过程中相反地扭曲。这与传统的“倾斜杠杆-手臂假说”相冲突，我们必须考虑结合新结构的存在的新机制。在体外滑动过程中，激活的细丝很容易切成小段。我们将这些颗粒的实际复制品图像与肌动蛋白Holmes模型的模拟图像进行了比较，发现肌球蛋白结合中断了肌动蛋白细丝螺旋结构的相位。众所周知，肌动蛋白细丝在扭力的作用下是脆弱的。可能的情况是，这种扭矩力可以作为滑动的内在和强制性的机械过程来施加。我们将提出肌动球蛋白滑动机制的新假说，该假说可能涉及到微观领域中所有观察到的现象。较少

This study aimed to capture the instantaneous structure of sliding actomyosin by quick-freeze deep-etch electron microscopy and to analyze the three-dimensional structural changes not only of crossbridges but also of actin filaments which had been postulated to work as simple track of myosin-motor movement. After completion of innovative methodology for 3-D reconstruction of replica specimens, we started the application of similar process to the reconstruction of bulky solid structure like negatively stained specimens. We also devised another methodology to predict the expected deep-etch replica image of the protein molecule, utilizing its atomic coordinates. The structure of myosin heads in rigor-complex matched pretty well to the model proposed by docking the crystal structure of actin and myosin S1. However, most of the crossbridges under sliding condition, unlike prediction, exhibited the configuration kinked to the opposite direction from that of ATP-bound ones. Since the observed … More structure appeared very similar to that of pPDM-crosslinked myosin/ADP complex, we compared quantitatively the surface profile of replica images with that of simulated model-images, utilizing new pattern-matching procedure. The result clearly confirmed that myosin head is oppositely kinked during sliding. This conflicts conventional "Tilting lever-arm hypothesis" and we must consider the new mechanism incorporating the existence of the novel structure.Actin filaments easily cut into small pieces during in vitro sliding. We compared the actual replica images of such particles with the images simulated from Holmes model of actin, and found that the phase of actin filaments' helical structure was interrupted by myosin binding. It is known that actin filaments are fragile by the application of a torque-force. The possibility is that such torque force may apply as an intrinsic and mandatory mechanical process for sliding. We would propose the new hypothesis of actomyosin sliding mechanism that might implicate all the observed phenomena in microscopic fields. Less