CRYSTALS OF 143 KDA BOVINE INTERPHOTORECEPTOR RETINOID BINDING PRO

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The 143 kDa human/mammalian interphotoreceptor retinoid-binding protein (IRBP) is unusual in that it has no homology with other retinoid-binding proteins. It consists of a single polypeptide composed of four homologous modules each ~300 amino acid residues. Most studies into its function have focused on the visual cycle trafficking of 11-cis retinal and all-trans retinol between the rods and pigment epithelium, as well as of fatty acids. The four module containing full-length mammalian protein has been crystallized for the first time, in the presence of a fatty acid ligand. The crystals show diffraction at room temperature at the in-house rotating anode X-ray generator, but disorders on cryo-cooling. After experimenting with a number of cryo-cooling agents, PEG 35K seems to offer most cryo-protection, in the presence of 20% glycerol. We have now grown the crystals from 18% 35K PEG. In addition to routine data collection, we propose also to experiment with the high pressure cryo-cooling method developed by Dr. Sol Gruner at CHESS. Dr. Gruner's group has agreed to assist us in these experiments.