MONOMOLECULAR MECHANICS AND MUTANT MYOSINS

单分子机制和突变肌球蛋白

基本信息

批准号：
6338661
负责人：
James Spudich
金额：
$ 20.03万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-07-01 至 2001-06-30
项目状态：
已结题

项目摘要

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.

描述（取自应用程序）：我们将解决与肌球蛋白功能有关的两个主要领域。首先，我们将对肌动蛋白和肌球蛋白进行动态测量，以揭示肌球蛋白构象的变化，这些变化是解释观察到的位移所需的大小所需的大小。通过单个肌球蛋白头和肌动蛋白寡聚物之间的复合物的荧光能形成肌球蛋白的构象变化将在ATPase周期中进行定量，从而导致S1肌动蛋白结合形式的X射线晶体结构，这是S1的X射线晶体，这是动力学循环中的临界状态。使用荧光共振能传递（FRET）的初步数据表明，肌球蛋白杠杆臂确实可以通过大于50度的ARC通过挥杆来充当机械放大器。我们建议使用FET方法进一步完善这些数据，该方法可以确定不同种群的肌球蛋白头构象，从而确定leer臂的最大摆动角度以及最大的最大步骤尺寸。将检查在ATP和各种ATP类似物存在下的构象状态的数量。 Vale实验室已经开发了一种定制的构建激光显微镜，可以在单分子水平上测量FRET，我们计划与他合作，对肌球蛋白电机进行此类测量。与罗杰·库克（Roger Cooke）的小组一起，我们将通过将各种探针放在分子中所选位点上的各种探针来衡量肌球蛋白头中构象变化的其他方面。在所有情况下，我们都将使用半胱氨酸光肌球蛋白构建体，这是一种功能性电动机，该电动机基本上没有半胱氨酸残基。选定的位点将更改为半胱氨酸残基，用于用探针直接标记。上述技术还将通过蛋白质的诱变来应用于在周期的各个状态下捕获的肌球蛋白头。例如，可以以这种方式研究导致无法水解结合ATP的突变变化。将创建一个F-肌动蛋白三聚体，用于结晶和表征，并具有有限的肌球蛋白运动结构域。 F-肌动蛋白和F-肌动蛋白具有肌球蛋白结合的原子结构对于理解基于肌球蛋白的运动至关重要。肌动蛋白单体不会激活肌球蛋白ATPase，肌动蛋白的丝状形式尚未结晶。一个主要的障碍是仅创建肌动蛋白细丝的核心部分，即肌动蛋白的三聚体，并以纯粹的形式隔离。突变方法将用于试图隔离这种物种。它将追求其激活肌球蛋白ATPase活性并与肌球蛋白头结合结晶的能力。我们承认这是一个极高的风险项目。但是，我们乐观的是，有些运气，我们可以实现这一目标，而回报将很高。