Abstract
Abstract: :
Crouch et al. (Photochem & Photobiol 56: 251-255, 1992) showed that interphotoreceptor retinoid binding protein (IRBP) has a remarkable but not yet understood ability to protect retinoids from oxidative/isomeric degradation. Toward understanding the mechanism of this protection we have recently elucidated a structural homology between IRBP, photosystem II D1 C-terminal processing protease, and the enoyl-CoA isomerase/hydratase family, suggesting a common fold used in diverse settings ranging from proteolysis, to fatty acid isomerization to retinoid transport (Loew and Gonzalez-Fernandez. Structure, in press). Purpose:To determine whether interactions between IRBP and retinoids are affected by mutations in the putative ligand-binding fold. Methods:We expressed in E. coli the second module of Xenopus IRBP as a soluble thioredoxin fusion protein (X2IRBP). To date, V186H, E217D, and E217H were created and purified. Each substitution was confirmed by mass spectrometry and protein concentration determined by amino-acid analysis. The stoichiometry and Kd of all-trans retinol binding to X2IRBP and its mutants were determined by fluorescence spectroscopy. The ability of the recombinant proteins to protect all-trans retinol from oxidative degradation was evaluated by monitoring the absorbance of the complex at 325 nm over time. Results:X2IRBP: N = 1.57 +/- 0.074; Kd = 0.11 +/- 0.042. This data is virtually identical to that reported by Loew et al. (Exp Eye Res 73: 257-264, 2001). E217H: N = 2.18 +/- 0.22; Kd = 0.08 +/- 0.06. E217D: N = 0.88 +/- 0.11; Kd = 0.11 +/- 0.10. V186H: N = 1.71 +/- 0.05; Kd = 0.03 +/- 0.04. When complexed to the mutants, retinol oxidation was 2.5 to 4.6 times faster than when bound to X2IRBP. Conclusion:These mutants demonstrate a slight change in number of binding sites and Kd and appear to be deficient in their ability to protect against oxidative degradation. This is in contrast to our previous data showing no functional difference when residues outside of the fold were mutated (Baer et al. Mol Vis. 4:30, 1998).
Keywords: 527 protein structure/function • 571 retinoids/retinoid binding proteins • 554 retina