SYNTHETIC AND BIOLOGICAL STUDIES OF NUCLEIC ACIDS

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The focus is on light transduction in vision. Specifically, we will study the dim light vertebrate photoreceptor, rhodopsin. Three specific long- range goals are proposed. I. Chemical Mechanism of the Transmission of the Signal (Light-catalyzed Retinal Isomerization) from the Seven Helical Bundle (the Membrane Domain) to the Two Extra-membranous Domains. A conformational change in the cytoplasmic domain occurs on illumination of rhodopsin. This change initiates all the biochemistry of the visual transduction. Our goal is to understand the precise chemical nature of this change. II. Mechanisms in Amplification of the Signal. Study of the binding of GDP-transducin to Metarhodopsin II, exchange of GDP by GTP and release of Talpha GTP. This will include detailed mapping of the contacts between rhodopsin and different forms of transducin. III. Biochemistry and Mechanisms of Adaptation to Light. Understanding adaptation is the major ultimate goal. The following overall processes leading to desensitization and termination of visual transduction are not understood: 1, selection of sites of single phosphorylation and/or polyphosphorylations of rhodopsin by rhodopsin kinase (RK); 2, mechanisms of interactions of phosphorylated rhodopsin with arrestin and possibly other proteins as yet unknown; 3, how do these protein/protein interactions pre-empt the binding sites and structures that are necessary for amplification of the signal (Metarhodopsin II- transducin interaction). Can we understand the accompanying conformational changes in the proteins participating in these processes? IV. Experimental Approaches. These would be interdisciplinary, spanning organic chemistry, biochemistry, molecular biology and biophysics (this in collaborations with Biophysical research groups). Approaches involve extensive mutagenesis in the synthetic rhodopsin gene, expression and purification of the expressed mutant proteins, chemical studies of the mutants including inter and intra-molecular crosslinking, using photosensitive and other bifunctional groups. Introduction of one or two cysteine residues at specific sites and their use as handles for chemical and biophysical (fluorescence, EPR) studies. Studies of the sites of the binding sites of RK, transducin and arrestin to Metarhodopsin. Manipulation as well as limitations of the phosphorylation sites in rhodopsin by extensive amino acid replacements, the role of single V/S multiple phosphorylations in arrestin binding. These studies will include on and off rates of binding of transducin, RK and arrestin to Metarhodopsin II and its phosphorylated forms.