Binding studies of tear lipocalin: the role of the conserved tryptophan inmaintaining structure, stability and ligand affinity

The principal lipid binding protein in tears, tear lipocalin (TL), binds th e spin-labeled analog of lauric acid and the fluorescent fatty acid analogs , DAUDA and 16-AP at one site. The native ligands of TL compete for this bi nding site. A fluorescent competitive binding assay revealed that apo-TL ha s a high affinity for phospholipids and stearic acid (K-i) of 1.2 mu M and 1.3 mu M, respectively, and much less affinity font cholesterol (K-i) of 15 .9 mu M For fatty acids, binding affinity correlated with the length of the hydrocarbon chain. TI, binds most strongly the least soluble lipids permit ting these lipids to exceed their maximum solubility in aqueous solution. T hese data implicate TL in solubilizing and transporting lipids in the tear film. Phenylalanine, tyrosine and cysteine were substituted for Trp 17, the only invariant residue throughout the lipocalin superfamily. Cysteine subs titution resulted in some loss of secondary structure, relaxation of aromat ic side chain rigidity, decreased binding affinity for DAUDA, and destabili zation of structure. Mutants of TL, W17Y, and W17F showed a higher binding affinity for DAUDA than wild-type TL. Comparison of the results of tryptoph an 17 substitution in lipocalin with those of tryptophan 19 substitution in beta-lactoglobulin revealed important differences in binding characteristi cs that reflect the functional heterogeneity within the lipocalin family. ( C) 1999 Elsevier Science B.V. All rights reserved.