Substructure and Communication in Myosin and Actin

肌球蛋白和肌动蛋白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）为肌肉收缩和非肌肉细胞中的许多运动和运输过程提供动力。肌动球蛋白相互作用的循环涉及这些蛋白质的弱结合和强结合复合物的形成。力和运动是在弱结合和强结合肌动球蛋白状态之间的过渡期间或之后产生的。这个项目的第一个目标是确定肌动蛋白上特定位点对这种转变的作用和贡献。为此，将在酵母肌动蛋白的N-末端和环21-29和92-103中进行突变工程化，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测定，光谱研究和生化测定，以澄清弱结合和强结合肌动球蛋白状态之间的过渡的结构决定因素。本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球蛋白收缩功能中的作用。这一目标将通过特定的化学交联反应肌动蛋白单体之间的细丝。这些反应将使相邻肌动蛋白上的亚结构域1和2之间的界面变性，从而允许在体外运动性和其他测定中测试肌动蛋白动力学的功能意义。突变酵母肌动蛋白将与半胱氨酸取代制备，使这种交联反应，并引入探针监测肌动蛋白的构象状态。这项工作的结果将提供一个描述的变化发生在肌球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的肌动球蛋白。这项工作也将阐明肌动蛋白的作用及其动力学特性的肌动球蛋白的生物学功能。 这些信息对于详细了解这种蛋白质复合物的运动和力产生的基本生物过程是必要的。 肌动蛋白和肌球蛋白相互作用的一般原则和它们相互作用的调节，这将在这项工作中阐明，应有助于了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生成任务。这些项目的工作将为博士后，研究生和本科生提供广泛的培训，涉及分子生物学，生物化学和生物物理学及其在蛋白质结构和功能研究中的应用。

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.