Functional cavity dimensions of tear lipocalin

Purpose. We calibrated the cavity of tear lipocalin with a series of fluore scent labeled lipids of increasing chain length and varying diameter. Methods. Cavity length was assessed with competitive fluorescent assays in which DAUDA was displaced from apo-tear lipocalin with ligands of increasin g carbon chain lengths from C12-C24. The concentrations of competitors that inhibit 50% of the binding of DAUDA (IC50) were compared. Functional diame ters of tear lipocalin and beta -lactoglobulin were estimated with fatty ac ids bearing fluorescent labels of various diameters. The cavity dimensions of other lipocalins were derived from their published crystal structure coo rdinates. Results. In tear lipocalin, the binding affinities of fatty acids increased up to a carbon chain length of 18 (22.5 Angstrom) but remained constant fr om C18-C24. The cavity length of other lipocalins in crystal form were simi lar to tear lipocalin in solution. Tear lipocalin showed decreased binding affinities with progressively increasing ring dimensions of the ligand. In contrast to beta -lactoglobulin and retinol binding protein, tear lipocalin bound DAUDA and cholesterol in the calyx. Neither tear lipocalin nor beta -lactoglobulin bound P646 in their respective cavities. The calculated inte r-sheet distances at the mouth of the crystallized lipocalins ranged from 1 6-22 Angstrom. Conclusions. Tear lipocalin is more promiscuous than beta -lactoglobulin or retinol binding protein because of a greater functional diameter. Differen ces in ligand specificity of the various lipocalins can not be explained si mply by variation in cavity length or the inter-sheet distances at the caly x mouths as determined by crystal structure. Other factors may influence li gand specificity such as size and/or dynamic motion of loops between the be ta strands.