STRUCTURE AND FUNCTION OF BACTERIORHODOPSIN

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The objective is to understand the function of bacteriorhodopsin (bR) at the level of chemistry and atomic structure. bR is an archetype of a transmembrane protein where many amino acids are directly involved in the chemical process of transmembrane transport. One aim is to achieve a preparative purification of bacterioospin protein produced from a PBR322 beta- galactosidase-bacterioospin fusion vector in E. coli. The chromophore retinal is to be incorporated to generate bacteriorhodopsin with the same properties as those of wild type bR. Many individual residues of bR will be altered by site- directed mutagenesis, and effects on function assessed by three quantitative assays of stoichiometry and efficiency by which the protein pumps protons in response to light. The structure of each mutated protein will be assessed quantitatively using electron diffraction up to 2.65 A resolution, that will allow clear definition of whether a mutational effect is chemical or is merely one that perturbs the folding or structure. Only through this synthesis can chemical role in the function be assigned. A second aim is to achieve a three-dimensional structure at high resolution for bR. Since electron diffraction methods proved increasingly complex at resolutions beyond 3.5 Angstroms in the membrane plane, and at lower resolutions for titled images 3-D crystals of bR are the basis for x-ray diffraction. bR from Halobacterium halobium will be solubilized in Triton X-100, purified in a sterol detergent by size exclusion, heterogeneity removed by isoelectric focusing in nonyl glucoside, and crystallized in a fourth detergent. Linking together of individually successful steps is aimed at exceeding the current crystal size-limited x-ray resolution of 5.5 Angstroms resolution. Electron microscopy will assist in solving the crystal structure. Binding site residues for heavy metal-containing labels already localized in the structure are to be located in the sequence using site-directed mutagenesis and electron diffraction. This will further define the folding path of sequence within the structure in membranes. Results will yield insights in fundamental processes of energy transduction and transmembrane signalling in biology. Similarities to mammalian rhodopsin and to transmembrane channeling receptors will illuminate processes in vision, neurochemistry and cell-cell regulation.