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

肌动蛋白丝的协同性及其功能意义：通过快速冷冻深蚀刻复制品电子显微镜进行结构分析

• 批准号: 14380312
• 负责人: KATAYAMA Eisaku
• 金额: $ 10.82万
• 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: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380312/
• 关键词: Quick-Freeze Electron Microscopy; 3-D Reconstruction; Image Simulation; Actomyosin Sliding Movement; Conformational Change; Reaction Intermediate; Tilting Lever-Arm Hypothesis; Actin Filament; 3次元像再構成法; コンピュータ・シミュレーション; アクトミオシン; in vitro滑り運動アッセ

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

本研究旨在利用速冻深刻蚀电子显微镜捕捉肌动球蛋白滑动的瞬时结构，并分析肌动蛋白的三维结构变化，不仅是交叉桥，而且是肌动蛋白运动的简单轨迹。在完成复制品标本三维重建的创新方法后，我们开始将类似的方法应用于负染色标本等笨重实体结构的重建。我们还设计了另一种方法来预测蛋白质分子的预期深蚀刻复制图像，利用其原子坐标。严密复合体中肌凝蛋白头部结构与肌动蛋白与肌凝蛋白S1晶体结构对接模型吻合较好。然而，与预测不同的是，滑移条件下的大多数交叉桥呈现出与atp结合桥相反方向的构型。由于观察到的…More结构与ppdm交联肌球蛋白/ADP复合物的结构非常相似，我们利用新的模式匹配程序，定量地比较了复制图像与模拟模型图像的表面轮廓。结果清楚地证实了肌凝蛋白头部在滑动过程中是反向弯曲的。这与传统的“倾斜杠杆臂假说”相矛盾，我们必须考虑包含新结构存在的新机制。肌动蛋白丝在体外滑动过程中很容易被切成小块。我们将这些颗粒的实际复制图像与肌动蛋白Holmes模型模拟的图像进行了比较，发现肌动蛋白丝的螺旋结构的相位被肌凝蛋白结合所中断。众所周知，肌动蛋白丝在扭矩作用下易碎。这种扭力可能作为滑动的内在和强制性的机械过程而起作用。我们将提出一种新的关于肌动球蛋白滑动机制的假说，这种假说可能涉及到所有在微观领域观察到的现象。少

项目成果

Kohji Ito, Taro Q.P.Uyeda, Y.Suzuki, Kazuo Sutoh, Keiichi Yamamoto: "Requirement of domain-domain interaction for conformational change and functional ATP hydrolysis in myosin"Journal of Biological Chemistry. 278. 31049-31057 (2003)

Kohji Ito、Taro Q.P.Uyeda、Y.Suzuki、Kazuo Sutoh、Keiichi Yamamoto：“肌球蛋白构象变化和功能性 ATP 水解的域间相互作用的要求”生物化学杂志。

M.Tominaga: "Oiwa Higher plant myosin XI moves processively on actin with 35 nm steps at high velocity"EMBO Journal. (印刷中). (2003)

M.Tominaga：“Oiwa 高等植物肌球蛋白 XI 以 35 nm 的速度在肌动蛋白上持续移动”EMBO 杂志（2003 年出版）。

Higher plant myosin XI moves processively on actin with 35 nm steps at high velocity

• DOI: 10.1093/emboj/cdg130
• 发表时间: 2003-03-17
• 期刊: EMBO JOURNAL
• 影响因子: 11.4
• 作者: Tominaga, M;Kojima, H;Oiwa, K
• 通讯作者: Oiwa, K

The motor domain determines the large step of myosin-V

• DOI: 10.1038/415192a
• 发表时间: 2002-01-10
• 期刊: NATURE
• 影响因子: 64.8
• 作者: Tanaka, H;Homma, K;Ikebe, M
• 通讯作者: Ikebe, M

Motor function of unconventional myosin.

• DOI: 10.1007/978-1-4419-9029-7_13
• 发表时间: 2003
• 期刊: Advances in experimental medicine and biology
• 作者: M. Ikebe;Akira Inoue;S. Nishikawa;K. Homma;Hiroto Tanaka;A. Iwane;E. Katayama;R. Ikebe;T. Yanagida