SYNTHETIC AND BIOLOGICAL STUDY OF NUCLEIC ACIDS

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I. BACTERIORHODOPSIN: Bacteriorhodopsin, the single protein of the differentiated purple membrane in Halobacterium halobium, transduces light energy to chemical energy by pumping protons from inside to outside of the cell. The purpose of this work is to understand the mechanism of proton translocation by this membrane protein. Does the mechanism involve a proton channel or conductance along a proton "wire"? The experimental approach would involve specific amino acid replacements in the protein in order to ask specific questions. Mutant proteins will be prepared by site specific mutagenesis of the bacteriorhodopsin gene followed by expression. They will be examined for the following properties: (1) refolding, binding of retinal and regeneration of bacteriorhodopsin-like chromophore; (2) reconstitution into vesicles and ability to pump protons; (3) biophysical (Fourier transform infrared laser Raman spectroscopy) studies of the effects of mutations on the protein structure, on interactions between retinal and the protein and on different steps in the photochemical cycle; and (4) possible correlations between effects on photochemical cycle and on proton translocation. II. BIOCHEMISTRY OF LIGHT-TRANSDUCTION IN ROD OUTER SEGMENTS IN VERTEBRATE RETINA; STRUCTURE-FUNCTION STUDIES ON RHODOPSIN. A major objective is to understand the dynamics of rhodopsin; the structural change on bleaching, the interaction with GTPase and the regulation of phosphorylation and dephosphorylation. Studies of rhodopsin at membrane level would involve delipidation, denaturation, refolding and reconstitution. A major aim would be the study of structure-function relationships by site-specific mutagenesis of the gene and expression of the mutated gene products. Total synthesis of the rhodopsin gene has been undertaken so as to facilitate completely unrestricted mutagenesis throughout the gene. For this purpose a suitable number of unique restriction sites have been introduced at appropriate positions along the gene. Structures of the rod outer segment proteins involved in light transduction (GTPase, cGMP phosphodiesterase, the Na channel) are also being investigated by methods of recombinant DNA.