STRUCTURE DETERMINATION OF THE NA/K-ATPASE

本项目的目的是研究Na/ k - atp酶的结构

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.