Regulation of Mammalian Class VI Myosin

哺乳动物 VI 类肌球蛋白的调节

• 批准号: 6677934
• 负责人: Mitsuo Ikebe
• 金额: $ 38.56万
• 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: 2003-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6677934
• 关键词: X ray crystallography; actins; adenosine triphosphate; calcium ion; conformation; cryoelectron microscopy; hydrolysis; microfilaments; myosins; phosphorylation; protein engineering; protein localization; protein structure function; recombinant DNA

DESCRIPTION (provided by applicant): The goal of the proposed project is to clarify the molecular mechanism of both function and regulation of mammalian class VI myosin. Our preliminary studies suggest that Ca 2+and phosphorylation by small G-protein down-stream protein kinases regulate the motor activity of myosin VI. We will first examine whether the specific phosphorylation of myosin VI occurs in cells, and then study the mechanism of Ca 2+ and/or phosphorylation mediated regulation of myosin VI motor activity. A recent study by others and us has revealed that class VI myosin is a processive motor that travels on actin filaments for a long distance without dissociating from actin. However, the mechanism by which myosin VI moves processively along actin filaments is not understood. We will address this problem by using various biophysical and electron microscopy techniques. The best approach to show the processive movement of myosin VI 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 VI on actin will be monitored by visualizing the movement of beads attached to myosin VI on actin filament. The conformational changes of myosin VI during the mechanical cycle will be studied by single molecule polarization TIRF microscopy that measures the angular change of myosin head. The overall structural change of myosin VI with 0.1 nm resolution will be monitored by X-ray solution scattering. The structure of the two-headed myosin VI on actin filament will be studied by 3D image reconstitution of the myosin VI decorated actin filaments with cryo-electron microscopy. We will also determine the structural motifs responsible for the processivity and reverse directionality of myosin VI by analyzing the motor properties of the variants in which each structural motif is changed by genetic engineering technology. In order to achieve this goal, we will use recombinant DNA technology to produce engineered myosin VI molecules. Particular regions of the myosin VI molecule that are hypothesized to be critical for the uniqueness and/or regulation of motor function will be modified and functionally expressed. The motor function will then be analyzed by enzymatic analysis, biophysical analysis and in vitro motility assay, ,with a particular emphasis on the single molecule assay system. The itemized specific aims are: 1. To determine the regulatory mechanisms of myosin VI motor function; 2) To define the structural changes of myosin VI during the ATP hydrolysis cycle; 3) To define the mechanism by which myosin VI moves processively along actin filaments; 4) To identify the molecular determinant of the directionality of myosin VI.

描述（由申请人提供）：拟议项目的目标是阐明哺乳动物 VI 类肌球蛋白的功能和调节的分子机制。我们的初步研究表明 Ca 2+ 和小 G 蛋白下游蛋白激酶的磷酸化调节肌球蛋白 VI 的运动活性。我们将首先检查细胞中是否发生肌球蛋白VI的特异性磷酸化，然后研究Ca 2+ 和/或磷酸化介导的肌球蛋白VI运动活性调节的机制。我们和其他人最近的一项研究表明，VI 类肌球蛋白是一种进行运动，可以在肌动蛋白丝上长距离移动而不与肌动蛋白分离。然而，肌球蛋白 VI 沿着肌动蛋白丝持续移动的机制尚不清楚。我们将通过使用各种生物物理和电子显微镜技术来解决这个问题。显示肌球蛋白 VI 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，即使用光阱纳米技术进行机械测量，以及通过全内反射（TIRF）显微镜直接观察运动。肌球蛋白 VI 在肌动蛋白上的旋转运动将通过观察附着在肌动蛋白丝上肌球蛋白 VI 上的珠子的运动来监测。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机械循环期间肌球蛋白 VI 的构象变化。肌球蛋白 VI 的整体结构变化以 0.1 nm 的分辨率通过 X 射线溶液散射进行监测。将通过冷冻电子显微镜对肌球蛋白 VI 修饰的肌动蛋白丝进行 3D 图像重建来研究肌动蛋白丝上双头肌球蛋白 VI 的结构。我们还将通过分析通过基因工程技术改变每个结构基序的变体的运动特性，确定负责肌球蛋白 VI 的持续性和反向性的结构基序。为了实现这一目标，我们将利用重组DNA技术来生产工程肌球蛋白VI分子。肌球蛋白 VI 分子中被假设对运动功能的独特性和/或调节至关重要的特定区域将被修饰和功能表达。然后通过酶分析、生物物理分析和体外运动测定来分析运动功能，特别强调单分子测定系统。详细的具体目标是： 1. 确定肌球蛋白 VI 运动功能的调节机制； 2) 明确肌球蛋白VI在ATP水解循环过程中的结构变化； 3) 定义肌球蛋白VI沿着肌动蛋白丝持续移动的机制； 4) 确定肌球蛋白VI方向性的分子决定因素。