Investigation of the Myosin ATPase Mechanism

描述(申请人提供)：本研究有两个主要目的， 两者都专注于确定基本肌球蛋白的关键成分的目标 马达机构。首先，很明显肌球蛋白在没有肌动蛋白的情况下可以 水解三磷酸腺苷，但随后捕获水解物。它是被假设的 当肌球蛋白结合时，有一条逃生通道(后门)打开 肌动蛋白。如果是这样的话，莫里斯博士应该能够用 逐渐变大的侧链改变肌球蛋白动力学，主要是通过 减缓磷酸盐的释放。这些突变体与肌动蛋白的相互作用 提供对肌球蛋白机制的基本见解。莫里斯医生？第二个目标 是定义产生以下两个极端的结构变化 肌球蛋白功能。以莫里斯医生为基础？初步数据，他假设 肌球蛋白V，以及可能的其他肌球蛋白家族成员，其动力学特征是 从根本上不同于常规肌球蛋白II。这些动力学差异 可以允许这些电机单独工作或少量工作。莫里斯医生已经 初步证据表明，非肌肉肌球蛋白IIB的功能更像肌球蛋白V 比肌肉的肌球蛋白II更高，但它的序列与 常规的平滑肌肌球蛋白。因此，嵌合体的产生在 而非肌肉IIB将允许他定义基础区域 肌球蛋白动力学周期的根本变化。酶促表达的研究进展 肌球蛋白的活性片段(S1样和重质肌球蛋白样片段)将 通过杆状病毒/SF9细胞系统完成。功能评估 表达的肌球蛋白将包括ATPase测定，酶的测定 动力学参数和体外运动性(肌动蛋白细丝通过 肌球蛋白)。将获得重组肌球蛋白的低分辨结构 通过对S1修饰的低温电子显微图像进行三维重建 肌动蛋白细丝，并通过X射线获得高分辨率结构 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.