IRBP--STRUCTURE/FUNCTION

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DESCRIPTION (Adapted from applicant's abstract): Interphotoreceptor retinoid-binding protein (IRBP) transports 11-cis and all-trans retinoids between the RPE and photoreceptors by solubilizing retinoids and enhancing the release of 11-cis retinal from the RPE. The long-term goal is to understand the mechanisms by which IRBP fulfills these tasks. IRBP consists of four homologous modules each containing two hydrophobic ligand-binding sites and a non-ligand-binding region that is a candidate docking site for an interphotoreceptor matrix or cell surface receptor. It is reasoned that through gene duplication the structure of the modules diverged, creating sites tailored for each of IRBP's physiological ligands. The working hypothesis is that through gene duplication and divergence the modules obtained binding sites tailored for different ligands as well as domains that interact with the matrix or cell surface. Three specific aims are proposed: (1) The hypothesis predicts that IRBP possesses two classes of modules, each preferentially binding different ligand sets. The ligand affinity and specificity of each module will be compared with that of the full-length IRBP and selected module combinations. This will be complemented by mutagenesis studies of a single module. The two binding sites within the module will be characterized through Arg to Gln and Trp to Phe substitutions. (2) The structural requirements for IRBP's function will be determined under physiological conditions. Using eyecup preparations, IRBP, the individual modules, selected module combinations and truncated modules will be assessed for ability to support rhodopsin regeneration, and the delivery and release of retinoids at the RPE apical surface. Full physiological activity of IRBP may require combinations of modules, and non-ligand-binding regions that could be receptor-docking sites. (3) The X-ray crystal structure of an individual IRBP module will be determined. The size of an individual IRBP module, the functional unit of IRBP, is ideal for X-ray crystallography. Preliminary studies indicate that crystals of IRBP module 2 will be of sufficient quality to solve the structure of an individual module. Insights gained from the crystal structure will suggest further functional studies for future work.