STRUCTURE DETERMINATION OF THE NA/K-ATPASE

本课题的目的是研究Na/K-ATP酶的结构， 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，在那里它发挥着重要作用， 维持细胞体积，神经和肌肉的兴奋性，以及 在肾脏和肠道中吸收。 这种酶是 常规用于控制心律失常的强心苷。 首先，将在2000年构建酶的三维模型。 约2.2nm分辨率，使用三种互补方法：a） 3-三维重建的质量分布从倾斜的意见， 负染色的结晶片将提供结构 关于从膜突出的分子域的信息。 为 Na/K-ATP酶，这些代表总质量的相当大的部分。 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关于 两个表面的形态。 c）电子显微镜和图像 对片材的冷冻水合制剂的处理将给出结果 与从（a）中获得的结果互补，因为它们将揭示结构 酶的跨膜部分的组织。 一个主要的努力将致力于生物化学的修改， Na/K-ATP酶及其脂质环境 这种酶会被 其多肽链和碳水化合物部分的受控消化。 对酶的消化形式的结构分析将定位其 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 超过目前的2.2nm。 其他物理方法，包括电子 衍射，圆二色性，和低角X射线散射，将是 用于补充电子显微镜研究。 最终目标是获得酶的三维结构， 分辨率约为1.0nm，这将足以解决结构 结构域如螺旋和对应于通道的构型。 最后，通过将这些结果与关于 序列中，各个结构域的结构可以与它们的作用相关 在运输过程中。

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.