Structure and Function of Myosin VI

肌球蛋白 VI 的结构和功能

• 批准号: 6604921
• 负责人: H Lee Sweeney
• 金额: $ 41.4万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6604921
• 关键词: Baculoviridae; Sf9 cell line; X ray crystallography; actins; calcium ion; calmodulin; cryoelectron microscopy; intracellular transport; microfilaments; myosins; phosphorylation; protein localization; protein protein interaction; protein structure function; recombinant proteins; tissue mosaicism

DESCRIPTION (provided by applicant): Myosin VI may be unique among the myosin family members in that it moves toward the pointed (-) end of the actin filament. Furthermore, like myosin V, it is a processive motor that moves via surprisingly large, but variable size, steps along an actin filament. This study has several goals that include, delineating the structural determinants of the reverse directionality, determining the features that allow the large step size as well as the processive movement, and determining the role that calcium plays in the regulation of the motor. This project will utilize in vitro expression and functional assays that will enable structure/function studies of recombinant myosin VI. Expression of enzymatically active fragments (single- and double-headed fragments) of myosin VI will be accomplished with the baculovirus/SF9 cell system. Functional evaluation of the expressed myosin will include ATPase measurements, determination of enzyme kinetic parameters and in vitro motility (translocation of actin filaments by myosin), including single molecule assays (collaboration with Dr. James Spudich). Low-resolution structures of the recombinant myosins bound to actin will be obtained via three-dimensional reconstructions of cryo-electron micrographs derived from decorated actin filaments (collaboration with Dr. Ron Milligan). High-resolution structures of myosin VI in the absence of actin will be obtained by X-ray crystallography (collaboration and subcontract with Dr. Anne Houdusse). The goals of this project will be realized by addressing the following specific aims: 1.) delineate the structural domain(s) in myosin VI that account for myosin VI directionality; 2.) determine which kinetic and structural features are necessary for processive movement and the large step size of myosin VI; and 3) delineate putative modes of regulation of the myosin VI motor.

描述（由申请人提供）：肌球蛋白VI在肌球蛋白家族成员中可能是独一无二的，因为它朝着肌动蛋白细丝的尖头（ - ）末端移动。此外，像肌球蛋白V一样，它是一种过程，它通过沿肌动蛋白细丝的较大但可变的尺寸移动。这项研究的几个目标包括描述反向方向性的结构决定因素，确定允许大型步骤大小和过程运动的特征，并确定钙在电动机调节中的作用。该项目将利用体外表达和功能测定法，以实现重组肌球蛋白VI的结构/功能研究。肌球蛋白VI的酶活性片段（单头和双头片段）的表达将使用杆状病毒/SF9细胞系统来完成。表达肌球蛋白的功能评估将包括ATPase测量，确定酶动力参数和体外运动性（肌球蛋白对肌动蛋白丝的易位），包括单分子测定（与James Spudich博士的协作）。重组肌球蛋白的低分辨率结构将通过源自装饰的肌动蛋白丝（与Ron Milligan博士的合作）的冷冻电子显微照片的三维重建获得。在没有肌动蛋白的情况下，肌球蛋白VI的高分辨率结构将通过X射线晶体学（与Anne Houdusse博士的协作和分包）获得。该项目的目标将通过解决以下特定目的来实现：1。）描述肌球蛋白VI中的结构域，以说明肌球蛋白VI方向性； 2.）确定用于进程运动以及肌球蛋白VI的较大步骤所必需的动力学和结构特征； 3）描述肌球蛋白VI电动机调节的假定模式。