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

本研究的目的是捕捉瞬时结构的滑动肌动球蛋白的速冻深蚀刻电子显微镜和分析的三维结构的变化，不仅横桥，而且肌动蛋白丝已被假定为工作的简单轨迹肌球蛋白运动。在完成了复制标本的3-D重建的创新方法之后，我们开始将类似的过程应用于重建庞大的固体结构，如负染色标本。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品图像，利用其原子坐标。严格复合物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的晶体结构提出的模型非常匹配。然而，在滑动条件下，与预测不同，大多数的横桥表现出的配置扭结的方向相反，从ATP绑定的。因为观察到 ...更多信息 结构与pPDM交联的肌球蛋白/ADP复合物非常相似，我们利用新的模式匹配方法，定量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，在滑动过程中，肌球蛋白头反向扭结。这与传统的“倾斜臂假说”相冲突，我们必须考虑新的机制，包括新结构的存在。我们将这种粒子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，发现肌动蛋白丝螺旋结构的相位被肌球蛋白结合所中断。已知肌动蛋白丝在施加扭矩力时是易碎的。可能的是，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。我们提出了一个新的肌动球蛋白滑动机制的假说，它可能涉及所有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