MONOMOLECULAR MECHANICS AND MUTANT MYOSINS

单分子机制和突变肌球蛋白

基本信息

批准号：
6100625
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：
1999
资助国家：
美国
起止时间：
1999-07-15 至 2000-06-30
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（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，其大小足以解释观察到的位移。 ATP酶循环期间肌球蛋白的构象变化将通过单个肌球蛋白头和肌动蛋白寡聚体之间复合物的荧光能量形成进行定量，从而产生肌动蛋白结合形式的S1的X射线晶体结构，这是动力学中的关键状态循环。使用荧光共振能量转移（FRET）的初步数据表明，肌球蛋白杠杆臂确实可以通过摆动大于 50 度的弧度来充当运动性机械放大器。我们建议使用 FET 方法进一步细化这一数据，该方法允许人们确定不同群体的肌球蛋白头部构象，从而确定斜视臂的最大摆动角度以及由此产生的一次动力冲程的最大步长。将检查 ATP 和各种 ATP 类似物存在下的构象状态数量。 Vale 实验室开发了一种定制激光显微镜，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此类测量。我们将与罗杰·库克的团队一起，通过将各种探针放置在分子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，我们都将使用我们的半胱氨酸轻肌球蛋白构建体，它是一种基本上不含半胱氨酸残基的功能性运动。选定的位点将更改为半胱氨酸残基，以便用探针直接标记。上述技术也将应用于通过蛋白质诱变而停滞在循环的各种状态的肌球蛋白头。例如，导致无法水解结合的 ATP 的突变变化可以通过这种方式进行研究。将创建 F-肌动蛋白三聚体，用于具有或不具有结合肌球蛋白运动域的结晶和表征。 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至关重要。肌动蛋白单体不会激活肌球蛋白 ATP 酶，丝状形式的肌动蛋白尚未结晶。一个主要障碍是仅产生肌动蛋白丝的核心部分（肌动蛋白三聚体），并以纯形式分离它。将使用突变方法来尝试分离这样的物种。我们将研究其激活肌球蛋白 ATP 酶活性以及在有或没有肌球蛋白头结合的情况下结晶的能力。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，而且回报会很高。