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的主要未解决的问题 基于运动性。利用新发展的低温原子结构方法， 力显微镜，它可以提供纳米分辨率与大分子 配合物无金属涂层，我们着重于三大方面的顺利 在这个项目中肌肉收缩。第一个目标是寻找 在两个孤立的细胞中预测的结构变化和重排 单个肌球蛋白分子和肌动球蛋白复合物，在条件下， 对应于每个主要生化（ATP水解）事件， 跨桥循环我们的目标是获得2 nm或更高波长的结构信息。 不使用平均方法，并阐明了交叉桥 分子水平上的循环。第二个目标是相互作用 平滑肌磷酸酶与肌球蛋白，其中结合化学计量 将被检查。这些研究可望提供一个结构基础 了解平滑肌中这种重要酶的功能 调控第三个目标是平滑肌的结构变薄 在收缩调节中起着关键作用的细丝，然而， 关于其结构的信息很少，特别是当 还存在其它调节组分，例如钙调蛋白和钙调蛋白。 这里要研究的问题对于我们理解 肌肉收缩，并没有得到解决，部分原因是 现有结构方法的局限性。这是第一次尝试 应用低温原子力显微镜方法， 生物学问题，目前的许多技术限制可以 规避，提供了一个独特的机会，探索基本的 老鼠的特性与成熟的平滑肌结合 生理学，这种新方法很好地准备使基本的 有助于肌肉收缩。

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.