STRUCTURE DETERMINATION OF THE NA/K-ATPASE

NA/K-ATP酶的结构测定

• 批准号: 3288066
• 负责人: MANIJEH MOHRAZ
• 金额: $ 10.49万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-06 至 1988-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288066
• 关键词: X ray crystallography; adenosinetriphosphatase; cell membrane; chemical structure function; circular dichroism; crystallization; electron microscopy; electron optics; enzyme model; enzyme reconstitution; enzyme structure; image processing; kidney; lipids; lyophilization

The aim of this project is to study the structure of Na/K-ATPase using, as the principal research tool, electron microscopy and image processing of its crystalline sheets. The Na/K-ATPase pump is found in the plasma membrane of most eukaryotic cells, where it plays a major role in maintenance of cell volume, excitability in nerve and muscle, and absorption in the kidney and intestine. The enzyme is the target of cardiac glycosides that are used routinely for controlling arrhythmias. Initially, a three-dimensional model of the enzyme will be constructed at about 2.2nm resolution using three complementary approaches: a) 3-dimensional reconstruction of the mass distribution from tilted views of the negatively stained crystalline sheets will provide structural information about molecular domains protruding from the membrane. For Na/K-ATPase, these represent a substantial fraction of the total mass. b) Freeze-drying and high resolution shadowing of the crystalline sheets and the use of surface reconstruction methods will supply information about the morphology of the two surfaces. c) Electron microscopy and image processing of frozen hydrated preparations of the sheets will give results complementary to those obtained from (a) as they will reveal the structural organization of the membrane-spanning part of the enzyme. A major effort will be devoted to the biochemical modification of Na/K-ATPase and its lipid environment. The enzyme will be altered by controlled digestion of its polypeptide chains and carbohydrate moiety. Structural analysis of the digested forms of the enzyme will locate its various domains. The lipid environment of the enzyme will be modified to improve the crystallinity of the sheets so as to extend the resolution beyond the current 2.2nm. Other physical methods, including electron diffraction, circular dichroism, and low angle X-ray scattering, will be employed to complement the electron microscopy studies. The ultimate goal is to obtain a 3-dimensional structure of the enzyme at a resolution of about 1.0nm, which would be sufficient to resolve structural domains such as helices and configurations corresponding to channels. Finally, by correlating these results with the information about the sequence, the structure of the various domains can be related to their role in the transport process.

该项目的目的是研究 Na/K-ATPase 的结构，例如 主要研究工具，电子显微镜和图像处理 它的结晶片。 Na/K-ATP酶泵存在于血浆中 大多数真核细胞的膜，在其中起重要作用 维持细胞体积、神经和肌肉的兴奋性，以及 在肾脏和肠道吸收。 酶的目标是 强心苷，通常用于控制心律失常。 最初，将在以下位置构建酶的三维模型： 使用三种互补方法约 2.2nm 分辨率：a) 从倾斜视图对质量分布进行 3 维重建 负染色的结晶片将提供结构 有关从膜突出的分子域的信息。 为了 Na/K-ATP酶，它们占总质量的很大一部分。 b) 结晶片的冷冻干燥和高分辨率阴影 表面重建方法的使用将提供有关 两个表面的形貌。 c) 电子显微镜和图像 对片材的冷冻水合制剂进行处理将给出结果 补充从（a）中获得的结果，因为它们将揭示结构 酶跨膜部分的组织。 将主要致力于生物化学修饰 Na/K-ATP酶及其脂质环境。 酶将被改变 其多肽链和碳水化合物部分的受控消化。 对酶的消化形式进行结构分析将定位其 各种领域。 酶的脂质环境将被改变 提高片材的结晶度，从而提高分辨率 超越目前的2.2nm。 其他物理方法，包括电子 衍射、圆二色性和小角度 X 射线散射 用于补充电子显微镜研究。 最终目标是获得酶的 3 维结构 分辨率约为1.0nm，足以解决结构问题 域，例如与通道对应的螺旋和构型。 最后，通过将这些结果与有关信息相关联 序列，各个域的结构可以与其作用相关 在运输过程中。