STUDIES OF VISUAL CYCLE PROTEINS

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The major emphasis of the proposed research is to elucidate the complete primary structure of the cellular retinaldehyde-binding protein (CRALBP) from bovine retina, a protein detected only in retina and retinal pigment epithelium (RPE) and which endogenously carries 11-cis-retinaldehyde and/or 11-cis-retinol. CRALBP could be a key component of the dark regeneration of 11-cis-retinaldehyde in the visual cycle and may also function as a substrate carrier protein for an 11-cis-retinol dehydrogenase found in RPE. Appropriate attention will be given to the identification of the NH2-terminal blocking group by fast atom bombardment-mass spectroscopy, to the identification possible sites of phosphorylation and to the evolutionary relatedness of CRALBP to other proteins. A concerted effort will also be made to crystallize CRALBP in a form suitable for X-ray crystallographic analysis. A long term goal is to obtain the three dimensional structure of the CRALBP 11-cis-retinaldehyde complex. The whole protein (size 33,000 daltons) will be fragmented by limited proteolysis to provide substructural domains which will be tested for residual retinoid-binding properties. The whole denatured protein will also be cleaved at asparaginyl-glycine, aspartyl-proline, methionyl, lysyl or arginyl bonds to generate fragments. The products of the various cleavage reactions will be purified and subjected to Edman degradation by automatic gas-phase and manual methodologies. Integration of these results is expected to provide the complete amino acid sequence. A coordinated approach will also be made to clone the gene for CRALBP and determine the cDNA structure. Met, Trp and Cys containing peptides will be selectively isolated and analyzed for their design potential for synthetic oligonucleotide hybridization probes. CRALBP antibodies will also be used for cDNA screening. Structural analysis will provide a precise molecular framework in which questions concerning the normal function of CRALBP and the visual disorders it may be associated with can be answered in specific terms.