Molecular mechanisms of myosin II-actin interactions

背景肌肉收缩是由分子运动肌球蛋白II与肌动蛋白的周期性相互作用驱动的。肌球蛋白与肌动蛋白结合后，ATP水解;磷酸盐（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燃料，同时发生分子结构的变化。肌球蛋白的一个小的构象变化产生所谓的“动力冲程”，并将肌动蛋白丝滑向肌节的中心。许多肌球蛋白马达排列成细丝，最终导致肌肉缩短。还有一些关键问题仍然未知，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）确定在动力冲程期间由肌球蛋白产生的力、ATP水解和Pi释放之间的关系（ii）确定肌球蛋白结构变化和动力冲程之间的关系（iii）确定肌球蛋白分子是否在细丝中协同工作（iv）确定肌原纤维中的肌节之间是否存在协同方法。我的实验室将使用各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）将通过测试具有不同结构的肌球蛋白分子来实现，这些结构改变了活性位点的Pi释放，使用新获得的激光陷阱系统，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可视化具有不同结构改变的肌球蛋白分子与肌动蛋白相互作用来实现，该显微镜允许在真实的时间内可视化肌球蛋白分子动力学。目标（iii）将通过肌球蛋白的双峰来实现，以检查它们在与动作相互作用期间是否相互影响，和肌球蛋白分子的细丝，同时与肌动蛋白Gola（iv）相互作用，将通过测试肌原纤维进行评估，肌原纤维由几个肌节串联组成，以检查一个肌节的激活或失活是否会改变相邻肌节的行为。意义肌球蛋白动力学和ATP动力学之间的关系是肌肉收缩中最基本的步骤。肌球蛋白分子的构象变化最终导致宏观尺度的收缩，并定义肌肉的功率输出。虽然这一提议是针对肌肉收缩的机制，但肌球蛋白只是许多具有类似结构的分子马达之一，它们沿着沿着生物路径行进，同时转换化学能以产生机械功。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、神经元发育等。

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.