Molecular mechanisms of myosin II-actin interactions

肌球蛋白 II-肌动蛋白相互作用的分子机制

• 批准号: RGPIN-2022-04770
• 负责人: Rassier, Dilson
• 金额: $ 4.74万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=757856
• 关键词: Molecular; mechanisms; myosin; II; actin

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.

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。肌凝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了动力，与分子结构的变化同时发生。肌凝蛋白的一个小构象变化产生了所谓的“强力冲程”，并使肌动蛋白丝向肌节中心滑动。分布在细丝中的许多肌球蛋白马达的作用最终导致肌肉缩短。有一些关键的问题仍然未知，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。本提案的具体目标是：(i)确定肌凝蛋白在力量冲程、ATP水解和Pi释放期间的力量产生之间的关系（ii）确定肌凝蛋白结构变化与力量冲程之间的关系（iii）确定肌凝蛋白分子是否在细丝中协同工作（iv）确定肌原纤维方法中肌节之间是否存在协作。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）将通过测试具有不同结构的肌球蛋白分子来实现，这些肌球蛋白分子改变了活性位点的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与肌动蛋白相互作用的具有不同结构变化的肌球蛋白分子来实现，该显微镜允许实时可视化肌球蛋白分子动力学。目标（iii）将通过肌球蛋白的双联体来实现，以检查它们在与动作相互作用过程中是否相互影响，而肌球蛋白分子在与肌动蛋白相互作用时的细丝（iv）将通过测试肌原纤维来评估。由一系列的几个肌节组成，以检查一个肌节的激活或失活是否会改变邻近肌节的行为。的意义。肌凝蛋白力量行程和ATP动力学之间的关系是肌肉收缩的最基本步骤。肌球蛋白分子的构象变化最终导致宏观尺度上的收缩，并决定肌肉的能量输出。虽然这一建议是针对肌肉收缩的机制，肌凝蛋白只是许多具有类似结构的分子马达中的一个，它们沿着生物路径移动，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、神经元发育等。