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Function of Small G-Protein Binding Myosin

小 G 蛋白结合肌球蛋白的功能

基本信息

批准号：
7216338
负责人：
Mitsuo Ikebe
金额：
$ 32.6万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-04-15 至 2009-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7216338
关键词：
ATP Hydrolysis ATP phosphohydrolase Actins Binding Biochemical Biological Assay Brain Cells Characteristics Class Collaborations Coupled Coupling Cryoelectron Microscopy Dissociation Electron Microscopy Elements Engineering Event Family Fluorescence Microscopy Genetic Engineering Goals Hand Head Image Imagery Kidney Kinesin Kinetics Light Lung Measurement Measures Mechanics Microfilaments Microscopy Microtubules Minus End of the Actin Filament Molecular Monitor Monomeric GTP-Binding Proteins Motion Motor Motor Activity Movement Myosin ATPase Myosin Type I Myosin Type V N-terminal Neck Phosphorylation Physiological Play Production Protein Binding Proteins Regulation Reporting Research Personnel Resolution Rho-associated kinase Roentgen Rays Role Rotation Series Signal Transduction Solutions Structure System Tail Takeda brand of pioglitazone hydrochloride Techniques Technology Testis Three-Dimensional Image Three-Dimensional Imaging Tissues Transportation Walkers Walking X ray diffraction analysis X-Ray Diffraction base cell motility coping insight interest member mutant myosin VI novel optical traps programs reconstitution rho single molecule

项目摘要

DESCRIPTION (provided by applicant): Myosin IX is a member of the diverse myosin superfamily and distributed in a variety of tissues, however, its physiological function is unclear. A most intriguing finding is that myosin IX is a single headed processive motor with reverse directionality. Furthermore, it contains Rho GAP domain in its tail and Ras binding domain in its head domain, suggesting its function as a motor protein carrying signaling function. However, myosin IX function and regulation at a molecular level are largely unknown. The goal of the proposed project is to clarify the molecular mechanism of function and regulation of myosin IX. First, we will study how the single-headed myosin IX can move processively on actin filaments. The best approach to show the processive movement of myosin IX is the use of single molecule analysis. We will employ two techniques, i.e., mechanical measurement with optical trap nanometry and direct visualization of the movement by total internal reflection (TIRF) microscopy. The rotational motion of myosin IX on actin will be monitored by visualizing the movement of bead attached myosin IX on actin filament. Second, the conformational changes of myosin IX during the mechanical cycle will be studied by single molecule polarization TIRF microscopy that measures the angular change of myosin head, X-ray solution scattering that can determine the overall structural changes of myosin IX with 0.1nm resolution, and 3D image reconstitution of the structure of actin filament decorated with myosin IX with cryo-electron microscopy. Third, the kinetic analysis of acto-myosin IX will be done. The cross-bridge cycling of myosin is closely related with the myosin's ATPase cycle, therefore, the analysis of each kinetic step of acto-myosin IX is anticipated to yield the critical information to understand the motor mechanism of myosin IX. Fourth, we will clarify the structural elements that determine the processivity and the reverse directionality of myosin IX. Our recent finding that Rho kinase activates myosin IX motor activity sheds a light to understanding the regulation of myosin IX. We will further define the regulatory mechanism of myosin IX at the molecular and the cellular levels. In order to achieve the goal, we plan to use recombinant DNA technology as a means of production of engineered myosin IX and its mutants. The purified engineered myosin IX will be subjected to functional analysis by various biophysical, biochemical and electron microscopy techniques with particular emphasis on a single molecule assay system. The proposed project will clarify the function and regulation of myosin IX, a unique member of myosin super family, thus provide a new insight into the mechanism of actin based motility.

说明(申请人提供)：肌球蛋白IX是肌球蛋白超家族中的一员，分布于多种组织中，但其生理功能尚不清楚。一个最有趣的发现是，肌球蛋白IX是一种具有反向的单头前进马达。此外，它在其尾部含有Rho Gap结构域，在其头部含有Ras结合域，这表明它具有携带信号功能的运动蛋白的功能。然而，肌球蛋白IX在分子水平上的功能和调节在很大程度上是未知的。该项目的目标是阐明肌球蛋白IX的功能和调控的分子机制。首先，我们将研究单头肌球蛋白IX如何在肌动蛋白细丝上进行连续运动。显示肌球蛋白IX进行性运动的最好方法是使用单分子分析。我们将使用两种技术，即光学陷阱纳米技术的机械测量和全内反射(TIRF)显微镜对运动的直接可视化。肌球蛋白IX在肌动蛋白上的旋转运动将通过可视化附着在肌球蛋白微丝上的肌球蛋白IX的运动来监测。其次，将通过单分子偏振TIRF显微镜测量肌球蛋白头部的角度变化来研究肌球蛋白IX在机械循环中的构象变化，X射线溶液散射可以以0.1 nm的分辨率确定肌球蛋白IX的整体结构变化，以及利用冷冻电子显微镜对肌球蛋白IX修饰的肌动蛋白细丝结构的三维图像重建。第三，对acto-myosin IX进行动力学分析。肌球蛋白的跨桥循环与肌球蛋白的ATPase循环密切相关，因此，对acto-myosin IX的每一个动力学步骤的分析有望为了解肌球蛋白IX的运动机制提供关键信息。第四，我们将阐明决定肌球蛋白IX的顺向性和逆方向性的结构元素。我们最近的研究发现，Rho激酶激活了肌球蛋白IX的运动活性，这为理解肌球蛋白IX的调控提供了线索。我们将在分子和细胞水平上进一步明确肌球蛋白IX的调控机制。为了实现这一目标，我们计划使用重组DNA技术作为生产工程肌球蛋白IX及其突变体的一种手段。纯化的工程化肌球蛋白IX将通过各种生物物理、生化和电子显微镜技术进行功能分析，特别是单分子检测系统。该项目将阐明肌球蛋白超家族中独特的成员肌球蛋白IX的功能和调控，从而为肌动蛋白的运动机制提供新的见解。