Energy Transduction in Myosin

描述（由申请人提供）：肌凝蛋白通过与肌动蛋白丝的相互作用产生力和运动的能力对许多生物过程至关重要，包括肌肉收缩、细胞分裂和细胞内运输。肌凝蛋白在其酶循环的各个阶段的原子水平结构为肌凝蛋白利用力产生的分子机制提供了框架。这些结构以及其他生化和结构数据表明，肌凝蛋白通过将核苷酸结合区域的小构象变化耦合到轻链结合区域的大摆动来产生力，而肌凝蛋白与肌动蛋白紧密结合。然而，关于肌球蛋白如何在其ATP酶周期中改变其对肌动蛋白的亲和力的结构细节，以及肌动蛋白结合如何激活ATP水解产物（ADP和磷酸盐）的解离，从而触发力的产生，缺乏信息。目前的假设是，分离肌动蛋白结合结构域的大间隙迅速改变构象，允许在磷酸盐释放和力产生之前与肌动蛋白结合。此外，假设核苷酸结合域的开关II区直接将构象变化偶联到杠杆臂上。肌凝蛋白V是一种非肌凝蛋白，具有独特的结构和生化特性，将被用作研究肌凝蛋白肌动蛋白和核苷酸结合区特定构象变化的模型。内在的和外在的荧光探针将被战略性地放置，以测量肌动蛋白，核苷酸结合和杠杆臂区域在肌球蛋白的酶循环中的构象变化。此外，瞬态动力学实验将用于将构象变化与肌动球蛋白atp酶循环中的特定生化步骤联系起来。我们将使用计算方法提出与实验数据一致的肌球蛋白atp酶循环的构象途径。通过整合计算和实验数据，我们将阐明肌凝蛋白产生力的结构机制的关键细节，并进一步了解与肌凝蛋白点突变相关的遗传疾病，如家族性肥厚性心肌病。公共卫生相关性：该项目的目标是确定肌凝蛋白如何将化学能转化为推动肌肉收缩过程的力量和运动。实验和计算生物物理工具的结合将用于定义肌动球蛋白V atp酶循环的结构途径，这将填补关于肌球蛋白如何在肌肉收缩中产生力量的关键空白。由于肌球蛋白的点突变与遗传性疾病如家族性肥厚性心肌病相关，阐明肌球蛋白能量转导的结构途径可能会提高我们对这些疾病的理解并导致未来的治疗。

DESCRIPTION (provided by applicant): The ability of myosin to generate force and motion through its interaction with actin filaments is essential to many biological processes including muscle contraction, cell division, and intracellular transport. The atomic level structures of myosin in various stages of its enzymatic cycle have provided a framework of the molecular mechanism of force generation utilized by myosin. These structures as well as other biochemical and structural data suggest that myosin generates force by coupling small conformational changes in the nucleotide-binding region to a large swing of the light-chain binding region while myosin is strongly bound to actin. However, there is a lack of information about the structural details of how myosin alters its affinity for actin throughout its ATPase cycle, and how actin-binding activates the dissociation of the products of ATP hydrolysis (ADP and phosphate), which triggers force production. The current proposal hypothesizes that the large cleft that separates the actin-binding domain changes conformation rapidly to allow binding to actin prior to phosphate release and force generation. Moreover, the switch II region in the nucleotide-binding domain is hypothesized to directly couple conformational changes to the lever arm. Myosin V, a non-muscle myosin that has unique structural and biochemical properties, will be used as a model to examine specific conformational changes in the actin- and nucleotide-binding regions of myosin. Intrinsic and extrinsic fluorescence probes will be strategically placed to measure conformational changes in the actin-, nucleotide-binding, and lever arm regions during the enzymatic cycle of myosin. In addition, transient kinetic experiments will be used to correlate the conformational changes with specific biochemical steps in the actomyosin ATPase cycle. We will use computational methods to propose a conformational pathway of the myosin ATPase cycle consistent with our experimental data. By integrating the computational and experimental data we will elucidate critical details about the structural mechanism of force generation in myosin and further our understanding of genetic diseases associated with point mutations in myosin, such as Familial Hypertrophic Cardiomyopathy. PUBLIC HEALTH RELEVANCE: The goal of this project is to determine how myosin converts chemical energy into force and motion to drive the process of muscle contraction. A combination of experimental and computational biophysical tools will be utilized to define the structural pathway of the actomyosin V ATPase cycle, which will fill in critical gaps in what is known about how myosin generates force in muscle contraction. Since point mutations in myosin are associated with genetic diseases such as Familial Hypertrophic Cardiomyopathy, elucidating the structural pathway for energy transduction in myosin may improve our understanding of and lead to future treatments for these diseases.