Molecular Dynamics of Muscle Contraction

描述(申请人提供)：本项目的目标是确定肌动球蛋白结构动力学在肌肉收缩机制中的作用。该项目之前的工作已经证明了EPR和光学光谱在检测溶液和皮肤肌肉纤维中肌球蛋白和肌动蛋白的运动方面的能力和互补性。这项工作将继续下去，重点是(1)使用肌动蛋白和肌球蛋白突变体获得新的定点定向光谱探针结合位点以及功能和结构扰动，(2)在瞬时生化条件下检测结构动力学，以及(3)我们的光谱测量与X射线结晶学和电子显微镜结构分析的直接关联。总的方法是检测肌球蛋白和肌动蛋白在彼此生理相互作用以及与ATP相互作用时的结构动力学，设计实验来测试和改进特定的分子模型来产生力。目的1重点发展改进的光谱方法研究肌球蛋白和肌动蛋白在溶液和肌纤维中的结构动力学。在剩下的目标中，这些方法将被用来测试关于结构动力学在从弱到强肌球蛋白结合的力产生转变中的功能作用的特定假说：在目标2中，将探针连接到溶液中的肌球蛋白上，以测试其与肌动蛋白和核苷酸相互作用过程中的结构动力学模型。在目标3中，将探针连接到溶液中的肌动蛋白上，以测试其在与肌球蛋白-核苷酸复合体相互作用期间的结构动力学模型。在目标4中，将探针连接到溶液中的肌动蛋白和肌球蛋白上，以直接探测肌动蛋白-肌球蛋白界面的结构动力学。在AIM中，5个探针被连接到皮肤肌肉纤维中的肌球蛋白上，以直接探测结构动力学与力产生的耦合。这些光谱研究将与X射线结晶学和低温电子显微镜的结构分析相结合，以提供三维结构和生理功能的分子动力学之间的联系。 这项拟议的研究汇集了从分子遗传学到生物化学再到生物物理学的强大技术组合，以解决肌肉收缩的分子机制。特别是，这项工作对了解肌肉功能至关重要，这项工作产生的技术已经被用来提供对肌肉功能障碍的分子洞察。更广泛地说，这个定义明确的系统作为研究分子动力学和相互作用在马达蛋白质中的作用的模型，我们正在开发的方法应该被证明在分析这一领域的广泛问题方面是有效的。

DESCRIPTION (provided by applicant): The goal of this project is to determine the role of actomyosin structural dynamics in the mechanism of muscle contraction. Previous work on this project has demonstrated the power and complementary nature of EPR and optical spectroscopy in detecting the motions of myosin and actin in solution and in skinned muscle fibers. This work will continue, with increased emphasis on (1) the use of actin and myosin mutants to obtain new site-directed spectroscopic probe attachment sites and functional and structural perturbations, (2) the detection of structural dynamics under transient biochemical conditions, and (3) direct correlation of our spectroscopic measurements with structural analysis by x-ray crystallography and electron microscopy. The overall approach is to detect the structural dynamics of myosin and actin during their physiological interactions with each other and with ATP, designing experiments to test and refine specific molecular models for force generation. Aim 1 focuses on the development of improved spectroscopic methods for studying structural dynamics of myosin and actin in solution and in muscle fibers. In the remaining aims, these methods will be used to test specific hypotheses about the functional role of structural dynamics in the force-generating transition from weak to strong actomyosin binding: In Aim 2, probes are attached to myosin in solution, to test models for its structural dynamics during interaction with actin and nucleotides. In Aim 3, probes are attached to actin in solution, to test models for its structural dynamics during interaction with myosin-nucleotide complexes. In Aim 4, probes are attached to both actin and myosin in solution, to probe directly the structural dynamics of the actin-myosin interface. In Aim 5 probes are attached to myosin in skinned muscle fibers, to probe directly the coupling of structural dynamics to force generation. These spectroscopic studies will be coordinated with structural analysis by x-ray crystallography and cryo-EM, in order to provide a connection between 3D structure and the molecular dynamics of physiological function. The proposed research brings together a powerful combination of techniques, from molecular genetics to biochemistry to biophysics, to solve the molecular mechanism of muscle contraction. In particular, this work is of fundamental importance for understanding muscle function, and the technology generated by this work is already being used to provide molecular insight into muscle malfunction. More generally, this well-defined system serves as a model for studying the role of molecular dynamics and interactions in motor proteins, and the approaches we are developing should prove effective in the analysis of a wide range of problems in this field.