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• W2032801984 abstract "Interphotoreceptor retinoid-binding protein (IRBP) appears to target and protect retinoids during the visual cycle. X-ray crystallographic studies had noted a betabetaalpha-spiral fold shared with crotonases and C-terminal protein transferases. The shallow cleft formed by the fold was assumed to represent the retinol-binding site. However, a second hydrophobic site consisting of a highly restricted cavity was more recently appreciated during in silico ligand-docking studies. In this study, the ligand-binding environment within the second module of Xenopus IRBP (X2IRBP) is defined.Pristine recombinant polypeptide corresponding to X2IRBP was expressed in a soluble form and purified to homogeneity without its fusion tag. Phenylalanine was substituted for tryptophan at each of the putative retinol-binding domains (W450F, hydrophobic cavity; W587F, shallow cleft). Binding of 11-cis and all-trans retinol were observed in titrations monitoring retinol fluorescence enhancement, quenching of tryptophan fluorescence, and energy transfer. The effect of oleic acid on retinol binding was also examined.A ligand-binding stoichiometry of approximately 1:1 was observed for 11-cis and all-trans with K(d) in the tens of nanomolar range. The substitution mutants showed little effect on retinol binding in titrations after fluorescence enhancement. However, the W450F and not the W587F mutant showed a markedly reduced capacity for fluorescence quenching for both 11-cis and all-trans retinol. Oleic acid inhibited the binding of 11-cis and all-trans retinol in an apparent noncompetitive manner.The binding site for 11-cis and all-trans retinol is a novel hydrophobic cavity that is highly restrictive and probably distinct from the long chain fatty acid-binding site." @default.
• W2032801984 created "2016-06-24" @default.
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• W2032801984 date "2009-12-01" @default.
• W2032801984 modified "2023-10-14" @default.
• W2032801984 title "Retinol-Binding Site in Interphotoreceptor Retinoid-Binding Protein (IRBP): A Novel Hydrophobic Cavity" @default.
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• W2032801984 doi "https://doi.org/10.1167/iovs.08-1857" @default.
• W2032801984 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5568800" @default.
• W2032801984 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19608538" @default.
• W2032801984 hasPublicationYear "2009" @default.
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