Substructure and Communication in Myosin and Actin

Reisler Myosin和actin是两种主要的肌肉蛋白，它们以循环的方式相互作用，并使用细胞的化学燃料三磷酸腺苷（ATP）来驱动肌肉收缩和非肌肉细胞的许多运动和运输过程。肌动球蛋白相互作用的循环涉及这些蛋白质的弱和强结合复合物的形成。力和运动是在弱和强结合的肌动球蛋白状态之间的过渡期间或之后产生的。该项目的第一个目标是确定肌动蛋白上的特定位点在这种转变中的作用和贡献。为此，将在酵母肌动蛋白的n端和环21-29和92-103上进行突变，以改变它们的电荷，并引入半胱氨酸残基用于荧光探针的附着。突变体和标记的突变体肌动蛋白将用于肌动球蛋白相互作用的体外运动分析、光谱研究和生化分析，以阐明弱和强结合肌动球蛋白状态之间转变的结构决定因素。该项目的第二个目标是阐明肌动蛋白丝的结构动力学在肌动凝蛋白收缩功能中的作用。这一目标将通过纤维中肌动蛋白单体之间的特定化学交联反应来实现。这些反应将固定相邻肌动蛋白上亚结构域1和2之间的界面，从而允许在体外运动和其他分析中测试肌动蛋白动力学的功能意义。突变酵母肌动蛋白将用半胱氨酸取代来制备，以实现这种交联反应，并引入用于监测肌动蛋白构象状态的探针。这项工作的结果将提供一个变化的描述，发生在肌凝蛋白和肌动蛋白之间的接触区域在不同步骤的力产生过程中由肌动蛋白。这一工作也将进一步阐明肌动蛋白及其动力学特性在肌动凝蛋白生物学功能中的作用。这些信息对于详细了解这种蛋白质复合物产生运动和力的基本生物过程是必要的。肌动蛋白和肌凝蛋白相互作用的一般原理及其相互作用的调控，将在这项工作中得到阐明，这将有助于理解其他蛋白质复合物如何在活细胞中执行类似的力和运动产生任务。这些项目的工作将为分子生物学、生物化学和生物物理学领域的博士后、研究生和本科生及其在蛋白质结构和功能研究中的应用提供广泛的培训。

9904599 Reisler Myosin and actin, the two major muscle proteins, interact with each other in a cyclic fashion and use the chemical fuel of cells, adenosine triphosphate (ATP), to power muscle contraction and many motile and transport processes in non-muscle cells. The cycle of actomyosin interaction involves the formation of weakly and strongly bound complexes of these proteins. Force and motion are generated during, or after, transition between the weakly and strongly bound actomyosin states. The first goal of this project is to determine the role and contribution of specific sites on actin to such a transition. To this end, mutations will be engineered in yeast actin, at its N-terminus, and loops 21-29 and 92-103, to alter their charge and to introduce cysteine residues for attachment of fluorescent probes. The mutant and labeled mutant actins will be used in the in vitro motility assays, spectroscopic studies, and biochemical assays of actomyosin interactions to clarify the structural determinants of the transition between weakly and strongly bound actomyosin states. The second goal of this project is to clarify the role of structural dynamics of actin filaments in the contractile function of actomyosin. This goal will be pursued through specific chemical cross-linking reactions between actin monomers in the filament. These reactions will immobilize the interface between subdomain 1 and 2 on adjacent actins, and thus allow the testing of functional significance of actin dynamics in the in vitro motility and other assays. Mutant yeast actins will be prepared with cysteine substitutions to enable such cross-linking reactions and to introduce probes for monitoring the conformational states of actin. The results of this work will provide a description of the changes which occur in the areas of contact between myosin and actin at different steps of the force generation process by actomyosin. This work will also clarify the role of actin and its dynamic properties in the biological function of actomyosin. Such information is necessary for a detailed understanding of the basic biological process of motion and force generation by this complex of proteins. General principles of actin and myosin interactions and the regulation of their interactions, which will be elucidated in this work, should help in the understanding of how other protein complexes carry out similar force and motion generation tasks in living cells. The work on these projects will provide broad training for postdoctoral, graduate, and undergraduate students in the areas of molecular biology, biochemistry, and biophysics and their applications in the studies on the structure and function of proteins.