The 11-cis isomer of vitamin A aldehyde is the chromophore of the visual pigment rhodopsin and can be regarded as being central to the entire visual process. This small molecule (MW 300) links, via a protonated Schiff base, with lysine 296 of the protein to form rhodopsin and, in this form, the chromophore holds the protein in an inactive conformation. Upon the absorption of a photon of light, the chromophore is isomerized to the all-trans isomer and induces conformational changes in the protein that place the protein in its active conformation. The apoprotein opsin, which does not contain the retinal and thus its """"""""lock"""""""" in the active or inactive configuration, has partial activity. It is proposed to use biochemical measures of rhodopsin kinase activity and physiological studies on intact photoreceptors in combination with analogs of retinal to explore these findings. This laboratory will probe the protein structure with mass spectral approaches utilizing crosslinking agents to examine the three-dimensional structure of both the active and inactive forms of the protein. The forms to be studied are: opsin (no chromophore; partially active), rhodopsin (11-cis retinal; inactive) and metarhodopsin II (all-trans retinal; active). Suggestions have been made that certain clinical disorders involving retinal degeneration may arise from prolonged activation of the transduction cascade. The fixing of the protein in its inactive or quiet state is likely to be essential to the normal physiological function of the retina. In addition, many steps in both the metabolism and transport of retinoids in the retina and RPE are ill-defined and warrant study. Using the retinal analogs developed here, the investigators propose to further explore the critical control of the activity of the rod and cone visual pigments and to use these analogs in examining a particularly perplexing protein in the RPE. Other projects include mapping of the chromophore-binding site and cytoplasmic loops of rhodopsin, and investigation of the role of the RPE-membrane protein, RPE-65.
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