Resolution of ligand positions by site-directed tryptophan fluorescence intear lipocalin

The lipocalin superfamily of proteins functions in the binding and transpor t of a variety of important hydrophobic molecules. Tear lipocalin is a prom iscuous lipid binding member of the family and serves as a paradigm to stud y the molecular determinants of ligand binding. Conserved regions in the li pocalins, such as the G strand and the F-G loop, may play an important role in ligand binding and delivery. We studied structural changes in the G str and of holo- and ape-tear lipocalin using spectroscopic methods including c ircular dichroism analysis and site-directed tryptophan fluorescence. Ape-t ear lipocalin shows the same general structural characteristics as hole-tea r lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progre ssive depth in the cavity from residues 105 to 99. For amino acid residues facing the internal aspect of cavity, the presence of a ligand is associate d with blue shifted spectra. The collisional rate constants indicate that t hese residues are not less exposed to solvent in hole-tear lipocalin than i n ape-tear lipocalin. Rather the spectral blue shifts may be accounted for by a ligand induced rigidity in holo-TL. Amino acid residues 94 and 95 are consistent with positions in the F-G loop and show greater exposure to solvent in the holo- than the ape-proteins. T hese findings are consistent with the general hypothesis that the F-G loop in the hole-proteins of the lipocalin family is available for receptor inte ractions and delivery of ligands to specific targets. Site-directed tryptop han fluorescence was used in combination with a nitroxide spin labeled fatt y acid analog to elucidate dynamic ligand interactions with specific amino acid residues. Collisional quenching constants of the nitroxide spin label provide evidence that at least three amino acids of the G strand residues i nteract with the ligand. Stern-Volmer plots are inconsistent with a ligand that is held in a static position in the calyx, but rather suggest that the ligand is in motion. The combination of site-directed tryptophan fluoresce nce with quenching by nitroxide labeled species has broad applicability in probing specific interactions in the solution structure of proteins and pro vides dynamic information that is not attainable by X-ray crystallography.