3-D CRYSTALLIZATION OF MEMBRANE PROTEINS

DESCRIPTION: The long range objectives of this research are to determine efficient methods for the formation of well ordered, 3-D crystals of Kdp ATPase from E. coli, cytochrome bd oxidase from E. coli, cytochrome aa3 oxidase from Rhodobacter sphaeroides, and cytochrome bo3 oxidase and complex II from E. coli. The Kdp ATPase is a member of the P-type ATPase family which includes the Na+/K+ - and Ca2+-ATPases. This enzyme provides an excellent system for determining the molecular basis for overall subunit organization, the nature of the K+ channel and the relationship between the site of phosphorylation and the ion channel, for example. The cytochrome oxidases from E. coli and Rb.sphaeroides are well studied members from the oxidase family of respiratory proteins. The selected proteins have been thoroughly characterized using biochemical, biophysical and genetic approaches and they are amenable to detailed structure/function studies. Complex II catalyzes the conversion of succinate to fumarate and carries out a key process in the tricarboxylic acid cycle and in bacterial and mitochondrial respiratory chains. The amino acid sequence of the multisubunit protein is highly conserved from bacteria to mammals and the E. coli complex is an ideal model system. The specific aims are to test new detergents for crystallization, to prepare Fv fragments which bind to tertiary epitopes on the membrane protein complexes, to carry out crystallization trials on the membrane proteins, alone and in combination with Fv domains, and to analyze the resulting crystals by x-ray diffraction. The Ferguson-Miller, Gennis, Kranz and Gouaux labs are expert in membrane protein/detergent interactions, in membrane protein overexpression and purification, in monoclonal antibody production, screening and Fv construction and expression, and in crystallization of membrane proteins and in x-ray crystallography, respectively. These goals will be accomplished by determining the thermal stability and biological function of the membrane proteins in the presence of new detergents (Ferguson-Miller), by isolating and purifying Fv fragments which bind to tertiary epitopes using standard immunological and molecular biological techniques (Kranz & Gennis) and by subjecting the proteins to sparse and systemic crystallization matrices designed for membrane proteins (Gouaux). On the basis of the proposed studies, general methods will be investigated and developed for 3-D crystallization of membrane proteins; for the proteins under study, crystals which are suitable for a high resolution structure determination may be obtained. Extending the methods elucidated from this research to other systems will provide a structural understanding of such human conditions as cystic fibrosis, retinitis pigmentosa and multidrug resistance.