We wish to understand the molecular basis of visual transduction. In particular, we intend to study (1) the key step in light to chemical energy conversion, the rhodopsin to bathorhodopsin transition, in two separate projects, (2) how the allosteric ligands, GDP and GTP, interact with transducin to better understand the chemical cascade reaction leading to cell hyperpolarization; and (3) the binding properties of retinoids to a class of retinoid binding proteins, some of which are involved in the visual cycle. In order to accomplish this goal, we propose several experiments employing state-of-the-art spectroscopic techniques which yield very detailed molecular information. Those used here are resonance Raman spectroscopy and quite novel non-resonance Raman ultra-sensitive difference spectroscopy as well as ultra-fast visible absorption and infrared kinetic spectroscopies. Four projects are proposed. We shall obtain the resonance Raman spectra of octopus rhodopsin and bathorhodopsin and isotopically labelled derivatives. This will be obtained in order to see if the molecular concepts developed for the well studied bovine system can be generalized to different species. Second, the kinetic steps involved in the rhodopsin to bathorhodopsin transition and aspects of the analogous primary process in bacteriorhodopsin, the bR568 to K photoreaction, will be studied by transient absorption spectroscopy (having a resolution of 0.1 ps) and transient infrared absorption spectroscopy (resolution of 0.5 to 1.0 ps). Thus, we shall be able to examine the kinetic pathway associated with the primary event of the chromophore's electronic changes as well as the vibrational steps of both the chromophore and the apoprotein . Thirdly, we shall obtain the Raman spectrum of GDP and GTP when bound to transducin, the G-protein of vision. The exchange of GDP for GTP at transducin's nucleotide binding pocket, which is catalyzed by excited rhodopsin, transforms transducin into an enzymatically active protein and is the first step in amplification of the light absorption event. In the fourth project, we shall employ pre-resonance Raman spectroscopy to study the binding of retinoids to three proteins (serum retinol binding protein, cytosolic retinol binding protein, and cellular retinaldehyde binding protein) which are specifically designed to bind these insoluble ligands. It is often suggested that the various retinoids are shuttled back and forth between the pigment epithelium and the photoreceptor cells by these type of soluble proteins but little is known how they bind the ligands.
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