Investigation of the Myosin ATPase Mechanism

肌球蛋白 ATP 酶机制的研究

• 批准号: 6340110
• 负责人: CARL A MORRIS
• 金额: $ 4.02万
• 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: 2002-02-15 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6340110
• 关键词: Baculoviridae; X ray crystallography; adenosine triphosphate; chemical kinetics; chimeric proteins; cryoelectron microscopy; crystallization; hydrolysis; model design /development; molecular genetics; molecular site; myosins; physical model; postdoctoral investigator; protein isoforms; protein purification; protein structure function; site directed mutagenesis; structural biology

DESCRIPTION (provided by applicant): There are two major aims of this study, both focused on the goal of characterizing key components of the basic myosin motor mechanism. First, it is clear that myosin in the absence of actin can hydrolyze ATP, but then traps the hydrolysis products. It has been hypothesized that there is an escape route (the back door) that opens when myosin binds to actin. If so, Dr. Morris should be able to block the back door with progressively large side chains that alter myosin kinetics, primarily by slowing phosphate release. The interaction of such mutants with actin will provide fundamental insights into the myosin mechanism. Dr. Morris? second aim is to define the structural alterations that generate the two extremes of myosin function. Based on Dr. Morris? preliminary data, he hypothesizes that myosin V, and likely other myosin family members, have kinetics that are fundamentally different from conventional myosin II. These kinetic differences may allow these motors to work either alone or in small numbers. Dr. Morris has preliminary evidence that nonmuscle myosin IIB functions more like myosin V than the myosin II of muscle, and yet it is 85% identical in sequence to conventional smooth muscle myosin. Thus, production of chimeras between smooth and nonmuscle IIB will allow him to define the regions that underlie fundamental changes in the myosin kinetic cycle. Expression of enzymatically active fragments (S1-like and heavy meromyosin-like fragments) of myosin 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). Low resolution structures of the recombinant myosins will be obtained via 3D reconstructions of cryo-electron micrographs derived from S1-decorated actin filaments, and high resolution structures will be obtained through X-ray crystallography.

描述（申请人提供）：这项研究有两个主要目的 两者都集中于表征基本肌球蛋白的关键成分的目标 电机机制。首先，很明显，在没有肌动蛋白的情况下肌球蛋白可以 水解ATP，但然后捕获水解产物。它已被假设 肌球蛋白结合到 肌动蛋白。如果是这样，莫里斯博士应该能够用 逐渐改变肌球蛋白动力学的大侧链，主要是 减慢磷酸盐的释放。这种突变体与肌动蛋白的相互作用将 提供有关肌球蛋白机制的基本见解。莫里斯博士？第二个目标 是定义产生两个极端的结构性改变 肌球蛋白功能。基于莫里斯博士？初步数据，他假设 肌球蛋白V，以及其他可能的肌球蛋白家庭成员，具有动力学 与常规肌球蛋白II根本不同。这些动力学差异 可以允许这些电动机单独或少量工作。莫里斯博士有 非肌肉肌球蛋白IIB功能更像肌球蛋白v的初步证据 比肌肉II的肌肉肌蛋白II，但依次是85％ 常规的平滑肌肌球蛋白。因此，在光滑之间产生嵌合体 Nonmuscle IIB将允许他定义基于的区域 肌球蛋白动力学周期的根本变化。酶的表达 肌球蛋白的活性碎片（类似S1的肌瘤状碎片）将 可以使用杆状病毒/SF9细胞系统完成。功能评估 表达的肌球蛋白将包括ATPase测量，确定酶 动力学参数和体外运动（通过 肌球蛋白）。将获得重组肌球蛋白的低分辨率结构 通过源自S1装饰的冷冻电子显微照片的3D重建 肌动蛋白丝和高分辨率结构将通过X射线获得 晶体学。