MONOMOLECULAR MECHANICS AND MUTANT MYOSINS

描述（摘自申请）：我们将讨论与肌球蛋白功能相关的两个主要领域。首先，我们将动态测量肌动蛋白和肌球蛋白，以揭示肌球蛋白的构象变化，所需的大小来解释所观察到的位移。在ATP酶循环过程中肌球蛋白的构象变化将使用荧光能量形成的一个单一的肌球蛋白头和肌动蛋白寡聚体之间的复合物进行定量，从而导致X射线晶体结构的肌动蛋白结合形式的S1，在动力学周期的临界状态。使用荧光共振能量转移（FRET）的初步数据表明，肌球蛋白杠杆臂可能确实作为一个机械放大器的运动，通过摆动超过50度的弧。我们建议进一步完善这一数据，使用FET的方法，使人们能够确定不同的人群的肌球蛋白头部构象，从而确定最大的摆角的杠杆臂和由此产生的最大步长的一个功率冲程。将检查在ATP和各种ATP类似物存在下的构象状态的数量。淡水河谷实验室已经开发出一种定制的激光显微镜，可以在单分子水平上测量FRET，我们计划与他合作，对肌球蛋白马达进行这样的测量。与罗杰库克的小组一起，我们将通过将各种探针放置在分子中的选定位点上来测量肌球蛋白头部构象变化的其他方面。在所有情况下，我们将使用我们的半胱氨酸-轻肌球蛋白构建体，这是一种基本上不含半胱氨酸残基的功能性马达。选择的位点将变为半胱氨酸残基，以便用探针直接标记。上述技术也将应用于通过蛋白质的诱变而停滞在周期的各种状态中的肌球蛋白头。例如，可以以这种方式研究导致不能水解结合的ATP的突变变化。将产生F-肌动蛋白三聚体用于结晶和表征，具有和不具有结合的肌球蛋白马达结构域。F-肌动蛋白和与肌球蛋白结合的F-肌动蛋白的原子结构对于理解基于肌球蛋白的运动是必不可少的。肌动蛋白单体不激活肌球蛋白ATP酶，肌动蛋白的丝状形式尚未结晶。一个主要的障碍是只产生肌动蛋白丝的核心部分，这是一个肌动蛋白三聚体，并以纯净的形式分离出来。将使用突变方法来尝试分离这样的物种。其激活肌球蛋白ATP酶活性的能力以及在肌球蛋白头部结合和不结合的情况下结晶的能力将被追求。我们承认这是一个风险极高的项目。然而，我们乐观地认为，如果运气好的话，我们可以实现这一目标，回报将很高。

Description (taken from the application): We will address two major areas relating to the function of myosin. First we will take dynamic measurements of actin and myosin to reveal changes in conformation of myosin that are of the size required to explain the observed displacements. Conformational changes in myosin during the ATPase cycle will be quantitated using fluorescence energy formation of a complex between a single myosin head and actin oligomers, leading to an X-ray crystal structure of the actin-bound form of S1, a critical state in the kinetic cycle. The preliminary data using fluorescence resonance energy transfer (FRET) suggests that the myosin lever arm may indeed function as a mechanical amplifier for motility by swinging through an arc greater than 50 degrees. We propose to further refine this data using FET approaches that allow one to ascertain different populations of myosin head conformations and thereby determine the maximum swing angle of the leer arm and the resultant maximum step size of one power stroke. The number of conformation states in the presence of ATP and various ATP analogs will be examined. The Vale laboratory has developed a custom build laser microscope that can measure FRET at the single molecule level, and we plan to collaborate with him to make such measurements for the myosin motor. Together with Roger Cooke's group, we will measure other aspects of conformational changes in the myosin head by placing various probes on chosen sites in the molecule. In all cases, we will use our cysteine-light myosin construct, which is a functional motor containing essentially no cysteine residues. Chosen sites will be changed to cysteine residues for direct labeling with probes. The above techniques will also be applied to myosin heads arrested in various states of the cycle via mutagenesis of the protein. For example, mutational changes that result in failure to hydrolyze bound ATP can be studied in this way. An F-actin trimer will be created for crystallization and characterization with and without bound myosin motor domain. Atomic structures of F- actin and F-actin with myosin bound are essential for understanding myosin-based motility. Actin monomers do not activate myosin ATPase and the filamentous form of actin has not been crystallized. A major hurdle is creating only the core part of the actin filament, which is an actin trimer, and isolating that in pure form. A mutational approach will be used to attempt to isolate such a species. Its ability to activate myosin ATPase activity and to crystallize with and without the myosin head bound will be pursued. We acknowledge that this is an extremely high risk project. We are optimistic, however, that with some luck we can achieve this goal, and the payoff will be high.