The overall objective of this proposal is to obtain a detailed description of the ultrastructure of (Na+, K+) ATPase and Ca2+ ATPase crystals induced by vanadate and other heavy metals in two conformational states (E1 less than greater than E2). Ca2+ ATPase crystals will be studied by freeze-fracture-etching, while (Na,K+) ATPase crystals will be analyzed by a combination freeze-fracture-etching, x-ray diffraction and 3-dimensional reconstruction of negatively stained and unstained preparations. The surface topology and substructure revealed on the fractured and etched surfaces by deep-etched rotary shadowing of the samples will be compared with the density map obtained with 3-D analysis of the negatively stained and unstained crystals. Complementary replicas will be used to determine the distribution of intramembranous particles and degree of penetration of protein molecules into lipid bilayers. The extent of plastic deformation and contamination during fracturing and replication will also be evaluated using complementary replicas. Comparison of density profiles by x-ray diffraction analysis of crystalline membranes and non-crystalline membranes where the protein to lipid ratio is low will provide direct information on how far the protein molecules extend from either side of the bilayer. Low angle reflections from the crystalline packing of the protein in the plane of the membrane will provide the dimensions of the crystal lattice. The position of wide-angle reflections on the x-ray films will provide the orientation of the alpha helices or beta sheets in the protein relative to the plane of the membrane. Three-dimensional analysis of negatively stained and unstained specimens provide complementary information that can be used to visualize much of the structure. The above studies should provide detailed information on the shape, molecular size, mass distribution, stoichiometry, localization and organization of (Na+, K+) ATPase and Ca2+ ATPase both within and on the surface of the membrane. The structural information should help us to understand the molecular mechanisms of these ion pumps.
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