CRYO ELECTRON MICROSCOPY OF ACTO-S1 ATPASE INTERMEDIATES

ACTO-S1 ATP酶中间体的冷冻电子显微镜

• 批准号: 2080364
• 负责人: HOWARD D. WHITE
• 金额: $ 19.77万
• 依托单位: EASTERN VIRGINIA MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2080364
• 关键词: actins; adenosinetriphosphatase; animal tissue; chemical binding; digital imaging; electron microscopy; enzyme mechanism; freezing; hydrolysis; muscle function; myosins; nucleoside triphosphate; protein purification; protein structure function; stop flow technique; structural biology; time resolved data

The molecular basis of muscle contraction is thought to be a configurational change in the myosin head while attached to actin. Such a change could either be a conformational change within the head itself, or it may be an alteration in the binding angle made with actin. The energy for force production Is known to come from ATP hydrolysis, but while the "tilting cross-bridge" hypothesis remains attractive, It is still unproven. Moreover, some recent data has suggested that the configuration of attached myosin heads may be similar in both the ATP and ADP states. This has added weight to suggestions that alternative force generation mechanisms should be considered. The main problem in testing the tilting bridge hypothesis has been the difficulty in establishing the structures of so-called weakly bound acto- myosin states that occur in the presence of ATP. A considerable amount is known about the structure of the strongly bound state that occurs in ADP or no nucleotide. However, weakly bound ATP states have been elusive because they dissociate at the low protein concentrations required for moderate resolution (approximately 3 nm) microscopy of the acto-myosin head (S1) complex. We recently found conditions that stabilize the weakly bound states and have been studying their structures by electron cryo- microscopy hydrated specimens. The micrographs already obtained show a variety of attached S1 configurations during steady- state ATP hydrolysis; such data are therefore compatible with the tilting bridge hypothesis but do not prove it. We now wish to extend this work to the study the structures of kinetically defined weak states in the ATPase cycle. If different kinetic states can be shown to have substantially different structures it will provide strong support for the tilting crossbridge hypothesis. This work will be facilitated by new methodology allowing time-resolved freezing of the electron microscope specimens to be carried out on the millisecond scale. The primary objective of this work is to obtain micrographs of biochemically defined weakly attached actomyosin states that are suitable for image processing methods. The long range goal is to obtain low resolution structures of weakly attached crossbridge states that can be used with the high resolutions crystal structures of actin and myosin to complement the actomyosin rigor structures that have been recently obtained. We believe this approach offers excellent prospects to resolve what has been arguably the single most important problem in understanding muscle function for more than two decades.

肌肉收缩的分子基础被认为是 附着在肌动蛋白上时肌球蛋白头部的构型变化。这样的 变化可能是头部本身的构象变化， 它可能是与肌动蛋白结合角度的改变。能量 力的产生是已知的来自ATP水解，但虽然 “倾斜的跨桥”假说仍然很有吸引力，它仍然是 未经证实此外，最近的一些数据表明， 在ATP和ADP状态下，附着的肌球蛋白头的可能是相似的。 这增加了关于替代部队生成的建议的份量， 应该考虑机制。 验证倾斜桥假说的主要问题是 在建立所谓的弱结合acto的结构中的困难， 肌球蛋白状态发生在ATP的存在。相当多的是 已知ADP中强束缚态的结构 或没有核苷酸。然而，弱结合ATP状态一直难以捉摸 因为它们在低蛋白浓度下解离， 中等分辨率（约3 nm）的肌动蛋白显微镜 头（S1）复合体。我们最近发现了一些条件， 结合态，并一直在研究它们的结构，通过电子冷冻， 显微镜水合标本。已经获得的显微照片显示， 在稳态ATP水解过程中，S1的各种连接构型; 因此，这些数据与倾斜桥假设是一致的， 不要证明它。 我们现在希望将这项工作扩展到研究动力学结构， 定义了ATP酶循环中的弱状态。如果不同的动力学状态 显示出具有实质上不同的结构，它将提供强有力的 支持了倾斜的横桥假说。这项工作将 新的方法，允许时间分辨冻结 电子显微镜标本进行毫秒级。 这项工作的主要目标是获得显微照片的 生物化学定义的弱附着肌动球蛋白状态， 用于图像处理方法。长期目标是获得低 解析结构的弱连接的跨桥状态，可以是 使用高分辨率的肌动蛋白和肌球蛋白晶体结构， 补充了肌动球蛋白的僵硬结构， 得到了我们认为，这一做法为解决这一问题提供了极好的前景。 在理解人类社会中， 超过20年的肌肉功能。