Molecular mechanisms of myosin II-actin interactions

背景资料。肌肉收缩是由分子马达肌球蛋白II与肌动蛋白的循环相互作用驱动的。肌球蛋白与肌动蛋白结合后，ATP被水解，磷酸(PI)和随后的ADP从肌球蛋白中释放出来。这些生化反应为肌球蛋白产生的机械功提供动力，与分子结构的变化同时发生。肌球蛋白的一个小的构象变化就会产生所谓的“强力中风”，并将肌动蛋白细丝滑向肌节的中心。许多排列在细丝中的肌球蛋白马达的动作最终会导致肌肉缩短。有一些关键问题仍然未知，并阻碍了我们对肌球蛋白-肌动蛋白相互作用的理解。我的研究计划的长期目标是了解肌肉收缩的分子机制。该建议的具体目标是：(I)确定肌球蛋白在力量卒中、ATP水解和PI释放期间产生的力之间的关系(Ii)确定肌球蛋白结构变化和力量卒中之间的关系(Iii)确定肌球蛋白分子是否在细丝中协同工作(Iv)以确定在肌原纤维方法中肌节之间是否存在协同作用。我的实验室将使用多种方法来测量肌球蛋白分子和细丝的力学和动力学。目标(Ii)将通过使用新获得的激光捕获系统来测试不同结构的肌球蛋白分子，这些分子改变了活性部位的PI释放，该系统能够以纳秒的时间分辨率测量PN尺度的分子力。目标(Ii)将通过使用高速原子力显微镜测试和显示具有不同结构变化的肌球蛋白分子与肌动蛋白相互作用来实现，从而允许实时可视化肌球蛋白分子动力学。目标(Iii)将通过双肌球蛋白来实现，以检查它们在与动作蛋白相互作用期间是否相互影响，以及(Iv)将通过测试由几个肌节组成的连续肌节组成的肌原纤维来评估肌球蛋白分子与肌球蛋白相互作用时的细丝，以检查一个肌节的激活或停用是否会改变相邻肌节的行为。意义重大。肌球蛋白动力和三磷酸腺苷动力学之间的关系是肌肉收缩最基本的步骤。肌球蛋白分子的构象变化最终导致宏观尺度上的收缩，并定义肌肉的功率输出。尽管这一提议针对的是肌肉收缩的机制，但肌球蛋白只是许多具有相似结构的分子马达之一，这些马达沿着生物路径移动，同时将化学能转化为机械功。分子马达是生命的引擎，负责细胞分裂、细胞迁移、组织生长、细胞内运输和神经元发育，仅举几例。

Background. Muscle contraction is driven by cyclical interactions of the molecular motor myosin II with actin. After myosin binds to actin, ATP is hydrolyzed; phosphate (Pi) and subsequently ADP are released from myosin. These biochemical reactions fuel the mechanical work produced by myosin, occurring simultaneously to changes in the structure of the molecule. A small conformational change in the myosin produces the so-called "power stroke" and sliding the actin filament towards the center of sarcomeres. The action of many myosin motors arranged  in filaments ultimately causes muscle shortening. There are critical issues that remain unknown and hamper our understanding of the myosin-actin interaction. The long-term goal of my research program is to understand the molecular mechanisms of muscle contraction. The specific goals of this proposal are: (i)To determine the relation between the force production by myosin during the power stroke, ATP hydrolysis, and Pi release (ii)To determine the relation between myosin structural changes and the power stroke (iii)To determine if myosin molecules work cooperatively in filaments (iv)To determine if there is cooperative among sarcomeres in a myofibril Methods. My laboratory will use a variety of methods to measure the mechanics and kinetics of myosin molecules and filaments. Goal (ii) will be achieved by testing myosin molecules with different structures that change Pi release form the active site, using a newly acquired laser-trap system with the capability of measuring molecular force in the pN scale with a time resolution of nanoseconds. Goal (ii) will be achieved by testing and visualizing myosin molecules with different structural alterations interacting with actin using a High-Speed Atomic Force Microscope, that allows the visualization of the myosin molecule dynamics in real time Goal (iii) will be achieved by doublets of myosins to check if they influence each other during interactions with action, and filaments of myosin molecules while interacting with actin Gola (iv) will be evaluated by testing myofibrils, comprised of several sarcomeres in series, to check if activation or deactivation of one sarcomere changes the behavior of adjacent sarcomeres. Significance. The relationship between the myosin power stroke and ATP kinetics is the most fundamental step in muscle contraction. Conformational changes in myosin molecules ultimately lead to contraction at the macro scale, and define the power output of muscles. Although this proposal is directed towards the mechanisms of muscle contraction, myosin is just one among many molecular motors with similar structures, which travel along biological paths while converting chemical energy to produce mechanical work. Molecular motors are the engines of life, responsible for cell division, cell migration, tissue growth, intracellular trafficking, neuronal development, to cite a few.