CRYO-ATOMIC FORCE MICROSCOPY (AFM) OF ACTOMYOSIN COMPLEXES

化学能转化为机械能的结构基础 运动是肌肉收缩和肌球蛋白尚未解决的主要问题 基于机动性的。利用最新发展的低温原子结构方法 力显微镜，它可以提供大分子的纳米分辨率 没有金属涂层的络合物，我们主要关注三个方面的流畅 在这个项目中肌肉收缩。第一个目标是寻找 预测的结构变化和重排都是孤立的 单个肌球蛋白分子和肌动球蛋白复合体 与每个重大生化(ATP水解)事件相对应 跨桥自行车。我们的目标是获得2纳米或2纳米的结构信息 更高，没有使用平均方法，并阐明了交叉桥 在分子水平上进行循环。第二个目标是 平滑肌磷酸酶与肌球蛋白结合的化学计量学 将会被检查。这些研究有望提供一个结构性的基础 为了了解这种重要的酶在平滑肌中的功能 监管。第三个目标是平滑肌层的结构变薄 细丝在收缩调节中起着关键作用，然而， 关于它的结构的信息很少，特别是当 其他调节成分，如钙调蛋白和钙桥蛋白，也存在。 这里要研究的问题一直是我们理解 肌肉收缩，一直没有得到解决，部分原因是 现有结构方法的局限性。这是第一次尝试 将低温原子力显微镜方法应用于定义明确的 生物问题，而目前的许多技术限制可以 提供了一个独特的机会来探索基本的 小鼠的特性。与成熟的平滑肌相结合 生理学，这种新的方法已经做好了准备，使基本 对肌肉收缩的贡献。

The structural basis for the conversion of chemical energy to mechanical movements is a major unresolved problem of muscle contraction and myosin based motility. Using the newly developed structural method of cryo atomic force microscopy, which can provide nm resolution with macromolecular complexes without metal coating, we focus on three major aspects of smooth muscle contraction in this project. The first objective is to search for the predicted structural changes and rearrangements in both isolated single myosin molecules and actomyosin complexes, under the conditions corresponding to each major biochemical (ATP hydrolysis) event during the crossbridge cycle. We aim at obtaining structural information at 2 nm or higher without using averaging methods, and elucidating the crossbridge cycle at the molecular level. The second objective is the interaction of smooth muscle phosphatase with myosin, where the binding stoichiometry will be examine. These studies are expected to provide a structural basis for understanding the function of this important enzyme in smooth muscle regulation. The third objective is the structure of smooth muscle thin filament which plays a pivotal role in the regulation of contraction, yet, very little information is available about its structure, especially when other regulatory components, such as caldesmon and calponin, are present. The problems to be studied here have been central for our understanding of muscle contraction, and have not been resolved, partly because of the limitations of available structural methods. This is the first attempt to apply the cryo atomic force microscopic method to a well defined biological problem, and many of the current technical limitations can be circumvented, providing a unique opportunity to explore the basic properties of mice. In combination with well established smooth muscle physiology, this new approach is well poised to make fundamental contributions to muscle contraction.