Function of Small G-Protein Binding Myosin

小 G 蛋白结合肌球蛋白的功能

• 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
• 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

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.

描述（申请人提供）：肌球蛋白IX是多种肌球蛋白超家族的成员，分布于多种组织中，但其生理功能尚不清楚。最有趣的发现是肌球蛋白 IX 是一种具有反向方向的单头处理马达。此外，它的尾部含有 Rho GAP 结构域，头部含有 Ras 结合结构域，表明其作为携带信号传导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 的功能和调节的分子机制。首先，我们将研究单头肌球蛋白 IX 如何在肌动蛋白丝上持续移动。显示肌球蛋白 IX 持续运动的最佳方法是使用单分子分析。我们将采用两种技术，即利用光阱纳米技术进行机械测量和通过全内反射 (TIRF) 显微镜直接观察运动。肌球蛋白 IX 在肌动蛋白上的旋转运动将通过观察肌动蛋白丝上附着的肌球蛋白 IX 珠的运动来监测。其次，将通过单分子偏振TIRF显微镜来研究肌球蛋白IX在机械循环过程中的构象变化，该显微镜测量肌球蛋白头部的角度变化，X射线溶液散射可以以0.1nm的分辨率确定肌球蛋白IX的整体结构变化，以及用冷冻电子显微镜重建肌球蛋白 IX 修饰的肌动蛋白丝结构的 3D 图像。第三，将进行肌动球蛋白IX的动力学分析。肌球蛋白的跨桥循环与肌球蛋白的ATP酶循环密切相关，因此，对肌球蛋白IX的每个动力学步骤的分析有望为理解肌球蛋白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 的持续性和反向性的结构元件。我们最近发现 Rho 激酶激活肌球蛋白 IX 运动活动，这为理解肌球蛋白 IX 的调节提供了线索。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技术作为工程化肌球蛋白IX及其突变体的生产手段。纯化的工程化肌球蛋白 IX 将通过各种生物物理、生物化学和电子显微镜技术进行功能分析，特别强调单分子测定系统。该项目将阐明肌球蛋白IX（肌球蛋白超家族的独特成员）的功能和调节，从而为基于肌动蛋白的运动机制提供新的见解。