G239T mutation in Repeat 1 of human IRBP: Possible implications for more than one binding site in a single repeat

PURPOSE: Interphotoreceptor retinoid-binding protein (IRBP) is a four-repea t protein found in the interphotoreceptor space. Each repeat can bind retin oids and fatty acids. The purpose of this study was to examine the effects of the single amino acid substitution, G239T, versus the wild type sequence of human IRBP Repeat 1, on ligand binding at equilibrium, ligand off rates , and protection of retinol from degradation. METHODS: G239T was created by site-specific mutagenesis, expressed in E. co li, and purified. E. coli expressed wild type Repeat 1 (EcR1) and G239T wer e subjected to thermal denaturation and analyzed by circular dichroism spec troscopy. We compared the ligand binding properties by fluorescence enhance ment of retinol and 16-anthroyloxy-palmitate, tryptophan quenching of the p roteins by different ligands, binding competition assays, protection of ret inol from degradation, and stopped-flow kinetics to measure transfer of lig ands to and from model membranes. RESULTS: Circular dichroism, fluorescence, and absorbance spectroscopy of G 239T and EcR1 showed similar wavelength scans. G239T exhibited about three- fold less fluorescence of bound all-trans-retinol or 13-cis-retinol versus EcR1. Retinol quenching of intrinsic protein fluorescence was reduced by 37 % in G239T versus EcR1. Other retinoids used as quenchers produced no diffe rence between intrinsic protein fluorescence of either G239T or EcR1; all e xhibited saturable high affinity binding to each protein. Docosahexaenoic a cid (DHA) served as a competitive inhibitor of retinol fluorescence enhance ment with EcR1. However, DHA did not alter retinol fluorescence with G239T. 16-anthroyloxy-palmitate (16-AP) exhibited about 30% higher levels of fluo rescence enhancement when bound to G239T versus EcR1. EcR1 prevented oxidat ive damage of all-trans-retinol, whereas G239T provided much less protectio n. Each protein could accept 9-cis-retinal from small unilamellar vesicles (SUVs) as measured by stopped flow kinetics. Off rates were the same in com paring G239T and EcR1 as acceptors. CONCLUSIONS: Despite the general similarity in shape between G239T and EcR1 and the nearly identical binding behavior with some ligands, distinct diff erences exist in the ligand binding properties of G239T and EcR1. Fluoresce nce enhancement/quenching and retinol protection experiments suggest that r etinol binding is reduced by about 50% in G239T versus EcR1. The data sugge st that either: (1) EcR1 contains two binding sites for retinol and G239T h as lost one site or (2) EcR1 has a single binding site that is altered in G 239T to reduce retinol binding. Results of all the experiments were consist ent with the first model while some of the data were not consistent with th e second model. Thus, it is possible that position 239, found in Domain B2 of IRBP Repeat 1, is located in or near one of two retinol binding sites.