CRYO-ATOMIC FORCE MICROSCOPY (AFM) OF ACTOMYOSIN COMPLEXES

肌动球蛋白复合物的低温原子力显微镜 (AFM)

• 批准号: 6494841
• 负责人: ZHIFENG SHAO
• 金额: $ 18.66万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6494841
• 关键词: actin binding protein; actins; atomic force microscopy; bioenergetics; bioimaging /biomedical imaging; biomedical equipment development; cell component structure /function; conformation; cryoscience; enzyme complex; enzyme structure; laboratory rabbit; microfilaments; muscle contraction; myosins; nanotechnology; phosphomonoesterases; phosphorylation; protein structure; smooth muscle; structural biology; vascular smooth muscle

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.

化学能转化为机械的结构基础 运动是肌肉收缩和肌球蛋白的主要尚未解决的问题 基于运动。使用新开发的冷冻原子结构方法 力显微镜，可以提供大分子的NM分辨率 没有金属涂料的复合​​物，我们专注于光滑的三个主要方面 该项目的肌肉收缩。第一个目标是搜索 孤立的预测结构变化和重排 在条件下 对应于每个主要的生化（ATP水解）事件 Crossbridge循环。我们旨在在2 nm或 不使用平均方法并阐明Crossbridge的更高 分子水平的循环。第二个目标是相互作用 肌球蛋白的平滑肌磷酸酶，其中结合化的化学计量法 将被检查。这些研究有望提供结构性基础 了解平滑肌中这种重要酶的功能 规定。第三个目标是平滑肌薄的结构 在收缩调节中起关键作用的细丝，但是 关于其结构的信息很少，尤其是 存在其他调节成分，例如Caldesmon和Calponin。 这里要研究的问题是我们理解的核心 肌肉收缩，尚未解决，部分原因是 可用结构方法的局限性。这是第一次尝试 将冷冻原子力显微镜方法应用于定义良好的 生物学问题，当前许多技术局限性可能是 规避，提供了一个独特的机会来探索基本 小鼠的特性。结合良好的平滑肌 生理学，这种新方法已经很好地制定了基本 对肌肉收缩的贡献。