Regulation of Mammalian Class VI Myosin

哺乳动物 VI 类肌球蛋白的调节

• 批准号: 6932297
• 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/6932297
• 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）：拟议项目的目的是阐明哺乳动物VI类肌球蛋白功能和调节的分子机制。我们的初步研究表明，小的G蛋白下游蛋白激酶的Ca 2+和磷酸化调节肌球蛋白VI的运动活性。我们将首先检查肌球蛋白VI的特异性磷酸化是否发生在细胞中，然后研究Ca 2+和/或磷酸化介导的肌球蛋白VI运动活性调节的机理。他人和美国的最新研究表明，VI级肌球蛋白是一种在肌动蛋白丝上长途旅行而不与肌动蛋白解离的方法。但是，尚不清楚肌球蛋白VI沿肌动蛋白丝进行过程移动的机制。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的过程运动的最佳方法是使用单分子分析。我们将采用两种技术，即具有光学陷阱纳米的机械测量，以及通过总内部反射（TIRF）显微镜直接可视化运动。肌球蛋白VI在肌动蛋白上的旋转运动将通过可视化肌动蛋白丝上附着在肌球蛋白VI上的珠的运动来监测。通过测量肌球蛋白头的角变化的单分子极化TIRF显微镜，将研究机械循环中肌球蛋白VI的构象变化。分辨率为0.1 nm的肌球蛋白VI的总体结构变化将通过X射线溶液散射监测。肌动蛋白丝上两头肌球蛋白VI的结构将通过3D图像重建的肌球蛋白VI装饰的肌动蛋白丝的3D图像重新构造，该肌动蛋白丝具有冷冻电子显微镜。我们还将通过分析基因工程技术更改每个结构基序的变体的运动性能，从而确定肌球蛋白VI的加工性和逆向方向性的结构基序。为了实现这一目标，我们将使用重组DNA技术来生产工程化的肌球蛋白VI分子。假设肌球蛋白VI分子的特定区域对于运动功能的唯一性和/或调节至关重要，将被修改并在功能上表达。然后，将通过酶学分析，生物物理分析和体外运动测定法对运动功能进行分析，并特别强调单分子测定系统。逐项特定的目的是：1。确定肌球蛋白VI运动功能的调节机制； 2）在ATP水解周期中定义肌球蛋白VI的结构变化； 3）定义肌球蛋白VI沿肌动蛋白丝进行过程的机制； 4）确定肌球蛋白VI方向性的分子决定因素。