Mechanochemistry of Myosin II Filaments

项目摘要 本项目将利用体外实验探索肌球蛋白II细丝在肌动蛋白细丝上运动的机械力化学。 方法：研究方法。我们将使用我们最近开发的反向运动分析来测量肌球蛋白细丝的速度。 在肌动蛋白细丝上移动。通过改变细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，告知肌肉收缩的机制。我们之前的工作揭示了如何组装 肌球蛋白进入细丝允许在生理数量的肌球蛋白在 细丝，与目前脱离受限动力学领域的范例形成对比。我们用这种化验方法 解决关于力和运动如何的具体结构和动力学假说的基础 肌球蛋白结合成细丝时产生的。肌球蛋白细丝，重组的和天然的，来自 我们将对平滑、骨骼、昆虫飞行和心肌进行比较。转基因果蝇将成为 产生来表达昆虫飞行肌肉肌球蛋白，具有缩短或延长的S2结构域，这是 肌球蛋白重链结构域，我们假设它是上述附着受限动力学的基础。 将检查肌球蛋白调节轻链的磷酸化的影响，目的是测试 关于肌球蛋白活性的新假说，它只有一个头部被磷酸化。化验结果将是 在两个方面进行了修改，以扩大其用途。首先，单个肌球蛋白头部将被标记上量子点和 并入到共丝中。运动中的细丝和量子点的整体运动 移动的细丝将同时可视化，并通过跟踪软件分析运动， 将评估预测的机械签名的存在或不存在。这些签名的身份将 通过与不断变化的实验条件的相关性进行评估，我们预测这些条件将改变签名。另外， 倒置的体外运动试验将被修改，以允许测量肌球蛋白细丝在负荷下移动， 允许检查负载对动力学的潜在影响，并允许比较 不同的肌球蛋白产生能量。最后，我们将在这些方法的基础上使用倒置运动学 钙和钙调节细丝-粗丝滑动机制的实验研究 肌球蛋白头部捆绑。

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.