Functional cavity dimensions of tear lipocalin

Abstract

Purpose. We calibrated the cavity of tear lipocalin with a series of fluorescent 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 increasing carbon chain lengths from C12-C24. The concentrations of competitors that inhibit 50% of the binding of DAUDA (IC 50) were compared. Functional diameters of tear lipocalin and ß-lactoglobulin were estimated with fatty acids bearing fluorescent labels of various diameters. The cavity dimensions of other lipocalins were derived from their published crystal structure coordinates. Results. In tear lipocalin, the binding affinities of fatty acids increased up to a carbon chain length of 18 (22.5 Å) but remained constant from C18-C24. The cavity length of other lipocalins in crystal form were similar to tear lipocalin in solution. Tear lipocalin showed decreased binding affinities with progressively increasing ring dimensions of the ligand. In contrast to ß-lactoglobulin and retinol binding protein, tear lipocalin bound DAUDA and cholesterol in the calyx. Neither tear lipocalin nor ß-lactoglobulin bound P646 in their respective cavities. The calculated inter-sheet distances at the mouth of the crystallized lipocalins ranged from 16–22Å. Conclusions. Tear lipocalin is more promiscuous than ß-lactoglobulin or retinol binding protein because of a greater functional diameter. Differences in ligand specificity of the various lipocalins can not be explained simply by variation in cavity length or the intersheet distances at the calyx mouths as determined by crystal structure. Other factors may influence ligand specificity such as size and/or dynamic motion of loops between the ß strands.