Investigation of the Myosin ATPase Mechanism

描述（由申请人提供）：本研究有两个主要目的， 两人都致力于研究基础肌球蛋白的关键成分， 电机机构首先，很明显，肌球蛋白在缺乏肌动蛋白的情况下， 水解ATP，但随后捕获水解产物。已经假设 当肌球蛋白结合到 肌动蛋白。如果是这样，莫里斯医生应该可以用 逐渐变大的侧链改变肌球蛋白动力学，主要通过 减缓磷酸盐释放这种突变体与肌动蛋白的相互作用将 为肌球蛋白的作用机制提供了基本的见解。莫里斯医生第二目标 是定义产生两个极端的结构变化， 肌球蛋白功能根据莫里斯医生的说法初步数据，他假设， 肌球蛋白V和可能的其他肌球蛋白家族成员具有动力学， 与传统的肌球蛋白II完全不同。这些动力学差异 可以允许这些电动机单独工作或少量工作。莫里斯医生 非肌肉肌球蛋白IIB功能更像肌球蛋白V的初步证据 比肌肉的肌球蛋白II，但它是85%相同的序列， 常规平滑肌肌球蛋白。因此，在光滑的细胞之间产生嵌合体， 而非肌肉的联合调查局能让他确定 肌球蛋白动力学周期的根本变化。酶促表达 肌球蛋白的活性片段（S1样和重肌球蛋白样片段）将 用杆状病毒/SF9细胞系统完成。的功能评定 所表达的肌球蛋白将包括ATP酶测量、酶活性测定、 动力学参数和体外运动性（肌动蛋白丝的移位， 肌球蛋白）。将获得重组肌球蛋白的低分辨率结构 通过来自S1修饰的低温电子显微照片的3D重建， 肌动蛋白丝和高分辨率的结构将通过X射线获得 结晶学

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.