Mechanochemistry of Myosin II Filaments

项目摘要 本计画将探讨肌球蛋白II细丝在肌动蛋白细丝上移动的机械化学， 方法.我们将使用我们最近开发的反向运动试验来测量肌球蛋白丝的速度 在肌动蛋白丝上移动通过改变肌丝中肌球蛋白头的数量和ATP浓度， 我们开发了一个确定性模型，描述了定义和限制细丝速率的参数， 细丝滑动告知肌肉收缩机制。我们之前的工作揭示了组装 肌球蛋白进入细丝允许在肌球蛋白的生理数量下的附着限制动力学。 细丝，与目前的范例在限制动力学领域。我们使用该测定作为 进一步解决关于力和运动如何被 当肌球蛋白结合到肌丝中时产生。肌球蛋白丝，包括重组的和天然的， 将比较平滑肌、骨骼肌、昆虫飞行和心肌。转基因果蝇将成为 产生表达具有缩短或延长的S2结构域的昆虫飞行肌肌球蛋白，这是 肌球蛋白重链结构域，我们假设为上述附着限制动力学的基础。 将检查肌球蛋白调节轻链磷酸化的影响，目的是测试肌球蛋白调节轻链的磷酸化水平。 提出了一个关于只有一个头部磷酸化的肌球蛋白活性的新假说。对试剂盒进行 通过两种方式进行修改以扩展其实用性。首先，将单个肌球蛋白头部用量子点标记， 并入到共丝中。运动的细丝和量子点的全局运动在 移动的细丝将同时可视化，并且通过跟踪软件对运动的分析， 将评估是否存在预测的机械特征。这些签名的身份将 通过与不断变化的实验条件的相关性来评估，我们预测这些实验条件会改变签名。还有， 倒置体外运动性测定将被修改以允许测量在负荷下运动的肌球蛋白丝， 允许检查负荷对动力学的潜在影响，并允许比较 不同的肌球蛋白来发电最后，我们将建立在这些方法的基础上，使用反向运动 检测钙对细丝-粗丝滑动调节的潜在机制， 肌球蛋白头部结合。

Project Summary This project will explore the mechanochemistry of myosin II filaments moving on actin filaments using in vitro methods. We will use our recently-developed inverted motility assay to measure myosin filament velocities moving on actin filaments. By varying the number of myosin heads in the filaments and the ATP concentration, we have developed a deterministic model describing the parameters that define and limit the rate of filament- filament sliding that inform mechanisms of muscle contraction. Our prior work revealed how assembling myosin into filaments allows for attachment-limited kinetics at physiological numbers of myosins in the filaments, in contrast to the current paradigm in the field of detachment-limited kinetics. We use this assay as the basis for addressing further specific structural and kinetic hypotheses about how force and motion are generated when myosin is incorporated into a filament. Myosin filaments, both reconstituted and native, from smooth, skeletal, insect flight, and cardiac muscle will be compared. Genetically engineered fruit flies will be generated to express insect flight muscle myosin with either shortened or lengthened S2 domains, which is the myosin heavy chain domain that we hypothesize to underlie the attachment-limited kinetics mentioned above. The effects of phosphorylation of the myosin regulatory light chains will be examined, with the goal of testing a novel hypothesis about the activity of myosin with only one of its heads phosphorylated. The assay will be modified in two ways to extend its utility. First, single myosin heads will be labeled with quantum dots an incorporated into co-filaments. The global motion of the moving filament and the quantum dot within the moving filament will simultaneously visualized, and by analysis of the motion by tracking software, the presence or absence of predicted mechanical signatures will be assessed. The identity of those signatures will be assessed by correlation to changing experimental conditions that we predict will change the signature. Also, the inverted in vitro motility assay will be modified to allow measurement of myosin filament moving under load, allowing underlying effects of load on kinetics to be examined and allow comparisons of the relative ability of different myosins to generate power. Final, we will build on these approaches by using the inverted motility assay to probe mechanisms underlying the regulation of thin filament –thick filament sliding by calcium and myosin head binding.