The distribution of lipids in tears is critical to their function. Lipids in human tears may retard evaporation by forming a surface barrier at the air interface. Lipids complexed with the major lipid binding protein in tears, tear lipocalin, reside in the bulk (aqueous) and may have functions unrelated to the surface. Many new lipids species have been revealed through recent mass spectrometric studies. Their association with lipid binding proteins has not been studied. Squalene, (O-acyl) omega-hydroxy fatty acids (OAHFA) and ceramides are examples. Even well-known lipids such as wax and cholesteryl esters are only presumed to be unbound because extracts of protein fractions of tears were devoid of these lipids. Our purpose was to determine by direct binding assays if the aforementioned lipids can bind tear lipocalin. Lipids were screened for ability to displace DAUDA from tear lipocalin in a fluorescence displacement assay. Di- and tri-glycerides, squalene, OAHFA, wax and cholesterol esters did not displace DAUDA from tear lipocalin. However, ceramides displaced DAUDA. Apparent dissociation constants for ceramide-tear lipocalin complexes using fluorescent analogs were measured consistently in the submicromolar range with 3 methods, linear spectral summation, high speed centrifugal precipitation and standard fluorescence assays. At the relatively small concentrations in tears, all ceramides were complexed to tear lipocalin. The lack of binding of di- and tri-glycerides, squalene, OAHFA, as well as wax and cholesterol esters to tear lipocalin is consonant with residence of these lipids near the air interface.

Phospholipid transfer protein, ∼80 kDa, transfers phospholipids from micelles to lipid binding proteins. The acceptor protein in plasma is apolipoprotein-A1, 28 kDa. Previously, phospholipid transfer protein was found in tears but an acceptor protein was not identified. To search for the acceptor protein(s) in tears a fluorescent phospholipid transfer assay was altered to omit the extrinsic acceptor. Human tears were incubated with fluorescent micelles and showed marked transfer activity verifying a native acceptor protein must be present. Reconstituted tears without tear lipocalin (lipocalin-1) eliminated the transfer of phospholipids. To determine if phospholipid transfer protein is involved in carrying phospholipid to the surface of tears from tear lipocalin, a fraction enriched in phospholipid transfer protein was injected into the subphase of a tear mimicking buffer in which tear lipocalin was present. The addition of phospholipid transfer protein did not increase the thickness of the surface layer regardless of the presence of lipid bearing tear lipocalin. The data show that phospholipid transfer protein transfers phospholipid from micelles to tear lipocalin. Phospholipid transfer protein does not transport the phospholipid. While tear lipocalin has no intrinsic transfer activity from micelles, it is the acceptor protein for phospholipid transfer protein in tears.

The stoichiometry of a ligand binding reaction to a protein is given by a parameter (n). The value of this parameter may indicate the presence of protein monomer or dimers in the binding complex. Members of the lipocalin superfamily show variation in the stoichiometry of binding to ligands. In some cases the stoichiometry parameter (n) has been variously reported for the same protein as mono- and multimerization of the complex. Prime examples include retinol binding protein, β lactoglobulin and tear lipocalin, also called lipocalin-1(LCN1). Recent work demonstrated the stoichiometric ratio for ceramide:tear lipocalin varied (range n = 0.3-0.75) by several different methods. The structure of ceramide raises the intriguing possibility of a lipocalin dimer complex with each lipocalin molecule attached to one of the two alkyl chains of ceramide. The stoichiometry of the ceramide-tear lipocalin binding complex was explored in detail using size exclusion chromatography and time resolved fluorescence anisotropy. Both methods showed consistent results that tear lipocalin remains monomeric when bound to ceramide. Delipidation experiments suggest the most likely explanation is that the low 'n' values result from prior occupancy of the binding sites by native ligands. Lipocalins such as tear lipocalin that have numerous binding partners are particularly prone to an underestimated apparent stoichiometry parameter.

Trp fluorescent spectra appear as a log-normal function but are usually analyzed with λmax, full width at half maximum, and the first moment of incomplete spectra. Log-normal analyses have successfully separated fluorescence contributions from some multi-Trp proteins but deviations were observed in single Trp proteins. The possibility that disparate rotamer environments might account for these deviations was explored by moment spectral analysis of single Trp mutants spanning the sequence of tear lipocalin as a model. The analysis required full width Trp spectra. Composite spectra were constructed using log-normal analysis to derive the inaccessible blue edge, and the experimentally obtained spectra for the remainder. First moments of the composite spectra reflected the site-resolved secondary structure. Second moments were most sensitive for spectral deviations. A novel parameter, derived from the difference of the second moments of composite and simulated log-normal spectra correlated with known multiple heterogeneous rotamer conformations. Buried and restricted side chains showed the most heterogeneity. Analyses applied to other proteins further validated the method. The rotamer heterogeneity values could be rationalized by known conformational properties of Trp residues and the distribution of nearby charged groups according to the internal Stark effect. Spectral heterogeneity fits the rotamer model but does not preclude other contributing factors. Spectral moment analysis of full width Trp emission spectra is accessible to most laboratories. The calculations are informative of protein structure and can be adapted to study dynamic processes.

Disulfide bonds play diverse structural and functional roles in proteins. In tear lipocalin (TL), the conserved sole disulfide bond regulates stability and ligand binding. Probing protein structure often involves thiol selective labeling for which removal of the disulfide bonds may be necessary. Loss of the disulfide bond may destabilize the protein so strategies to retain the native state are needed. Several approaches were tested to regain the native conformational state in the disulfide-less protein. These included the addition of trimethylamine N-oxide (TMAO) and the substitution of the Cys residues of disulfide bond with residues that can either form a potential salt bridge or others that can create a hydrophobic interaction. TMAO stabilized the protein relaxed by removal of the disulfide bond. In the disulfide-less mutants of TL, 1.0M TMAO increased the free energy change (ΔG(0)) significantly from 2.1 to 3.8kcal/mol. Moderate recovery was observed for the ligand binding tested with NBD-cholesterol. Because the disulfide bond of TL is solvent exposed, the substitution of the disulfide bond with a potential salt bridge or hydrophobic interaction did not stabilize the protein. This approach should work for buried disulfide bonds. However, for proteins with solvent exposed disulfide bonds, the use of TMAO may be an excellent strategy to restore the native conformational states in disulfide-less analogs of the proteins.