Regulation of Mammalian Class VI Myosin

项目描述（由申请人提供）：拟建项目的目标是阐明哺乳动物VI类肌球蛋白的功能和调控的分子机制。我们的初步研究表明ca2 +和小g蛋白下游蛋白激酶的磷酸化调节了myosin VI的运动活性。我们将首先研究细胞中是否发生了myosin VI的特异性磷酸化，然后研究ca2 +和/或磷酸化介导的myosin VI运动活性的调节机制。最近的一项研究表明，VI类肌凝蛋白是一种不与肌动蛋白分离而在肌动蛋白丝上长距离移动的过程马达。然而，肌凝蛋白VI沿着肌动蛋白丝前进的机制尚不清楚。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。单分子分析是显示肌凝蛋白VI过程运动的最佳方法。我们将采用两种技术，即用光学陷阱纳米测量法进行机械测量，以及用全内反射（TIRF）显微镜直接观察运动。肌凝蛋白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的肌凝蛋白VI上的小珠的运动来监测。通过测量肌球蛋白头部角度变化的单分子极化TIRF显微镜，研究了机械循环过程中肌球蛋白VI的构象变化。用x射线溶液散射法监测0.1 nm分辨率下肌球蛋白VI的整体结构变化。利用冷冻电镜对肌动蛋白修饰的肌动蛋白丝进行三维图像重建，研究肌动蛋白丝上的双头肌动蛋白VI的结构。我们还将通过分析每个结构基序通过基因工程技术改变的变体的运动特性来确定负责肌球蛋白VI的加工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技术生产工程肌球蛋白VI分子。肌凝蛋白VI分子的特定区域被假设为对运动功能的独特性和/或调节至关重要，这些区域将被修改和功能性表达。运动功能将通过酶分析、生物物理分析和体外运动分析进行分析，并特别强调单分子分析系统。具体目标是：1。探讨肌球蛋白VI运动功能的调控机制；2)明确肌球蛋白VI在ATP水解周期中的结构变化；3)确定肌凝蛋白VI沿肌动蛋白丝行进的机制；4)确定肌凝蛋白VI定向性的分子决定因素。

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.