PURPOSE: To study binding of retinoids to human tear lipocalin (TL) to assess factors influencing ligand affinity and delivery. Mechanistic features of retinoid interactions with TL were investigated, including the influence of the retinoid functional group on ligand affinity, the relative affinity of retinol versus fatty acids, the influence of relaxation of secondary structure in TL on ligand binding, the role of specific conserved hydrophobic residues in maintaining the rigidity of the secondary structure, and the potential release of retinol in a low-pH environment that promotes structural relaxation at lipid interfaces. METHODS: The binding and displacement of retinoids were monitored by quenching of protein fluorescence. Circular dichroic spectra were used to evaluate structural and conformational changes in TL-retinoid complexes. Site-directed mutagenesis was performed to determine the influence of the residues Trp17, Ile98, Gly15, and Leu19 in retinoid binding to TL and to correlate these effects with changes in secondary structure. RESULTS: Retinal and retinol bound TL with similar affinity. Fatty acids competed with retinoids for the same binding site on TL. Optical activity associated with retinal binding to TL was reduced in the presence of palmitic acid. In comparison with TL, the mutants W17C and I98C displayed relaxation of secondary structure, manifested as diminution of beta-sheet content in conjunction with a destabilization in urea, reduced aromatic asymmetry, and greater binding affinity for retinoids. Unlike fatty acids, retinol is not released from TL at low pH. CONCLUSIONS: The unique spectral properties of retinoids permit the simultaneous study of structural changes in TL and ligand binding. Retinoid binding is enhanced by specific mutations that induce relaxation of TL structure but is altered minimally by the functional group in retinoids. Two key hydrophobic residues, Trp17 (A strand) and Ile98 (G strand), contribute to backbone rigidity and influence retinoid binding through their participation in an internal hydrophobic cluster and external hydrophobic patch, respectively. The contributions of these sites to ligand binding may explain their conserved nature in the lipocalin family. Information regarding the binding and release of retinoids compared with fatty acids favors a role for TL in the delivery of lipids other than retinol to the tear film interfaces.
PURPOSE: To study binding of retinoids to human tear lipocalin (TL) to assess factors influencing ligand affinity and delivery. Mechanistic features of retinoid interactions with TL were investigated, including the influence of the retinoid functional group on ligand affinity, the relative affinity of retinol versus fatty acids, the influence of relaxation of secondary structure in TL on ligand binding, the role of specific conserved hydrophobic residues in maintaining the rigidity of the secondary structure, and the potential release of retinol in a low-pH environment that promotes structural relaxation at lipid interfaces. METHODS: The binding and displacement of retinoids were monitored by quenching of protein fluorescence. Circular dichroic spectra were used to evaluate structural and conformational changes in TL-retinoid complexes. Site-directed mutagenesis was performed to determine the influence of the residues Trp17, Ile98, Gly15, and Leu19 in retinoid binding to TL and to correlate these effects with changes in secondary structure. RESULTS: Retinal and retinol bound TL with similar affinity. Fatty acids competed with retinoids for the same binding site on TL. Optical activity associated with retinal binding to TL was reduced in the presence of palmitic acid. In comparison with TL, the mutants W17C and I98C displayed relaxation of secondary structure, manifested as diminution of beta-sheet content in conjunction with a destabilization in urea, reduced aromatic asymmetry, and greater binding affinity for retinoids. Unlike fatty acids, retinol is not released from TL at low pH. CONCLUSIONS: The unique spectral properties of retinoids permit the simultaneous study of structural changes in TL and ligand binding. Retinoid binding is enhanced by specific mutations that induce relaxation of TL structure but is altered minimally by the functional group in retinoids. Two key hydrophobic residues, Trp17 (A strand) and Ile98 (G strand), contribute to backbone rigidity and influence retinoid binding through their participation in an internal hydrophobic cluster and external hydrophobic patch, respectively. The contributions of these sites to ligand binding may explain their conserved nature in the lipocalin family. Information regarding the binding and release of retinoids compared with fatty acids favors a role for TL in the delivery of lipids other than retinol to the tear film interfaces.