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

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