Membrane Partitioning of TEMPO Discriminates Human Lung Cancer from Neighboring Normal Cells.

The plasma membranes of normal and cancer cells of the lung, breast, and colon tissues show considerably different lipid compositions that greatly influence their physicochemical properties. Partitioning of the spin probe 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) into the membranes of human lung normal and carcinoma cells was assessed by EPR spectroscopy to estimate the impact of the lipid compositions. The goal was to reveal potential strategies for cancer therapy attributable to the membrane properties. The study was conducted at pH values of 7.3 and 6.2, relevant to the microenvironments of normal and cancer cells, respectively. The TEMPO partitioning was examined in the temperature interval of 283-317K to reveal the efficacy of local hyperthermia used in chemotherapy. Results indicate that the TEMPO partitioning coefficient for the membranes of human lung carcinoma cells is significantly higher compared with that of neighboring normal cells. Increased partition coefficients were observed at relatively higher temperatures in both normal and cancer cells. However, compared to the normal cells, the cancer cells demonstrated higher partition coefficients in the studied temperature range. The data obtained with C12SL (spin-labeled analog of lauric acid) indicate that increased membrane dynamics of the cancer cells is a possible mechanism for enhanced partitioning of TEMPO. Free energy values for partitioning estimated for pH values of 6.2 and 7.3 show that TEMPO partitioning requires 30% less energy in the cancer cells at pH 7.3. TEMPO and its derivatives have previously been considered as theranostic agents in cancer research. Data suggest that TEMPO derivatives could be used to test if complementary alkalization therapy is effective for cancer patients receiving standard chemotherapy with local hyperthermia.

Site-directed tryptophan fluorescence reveals the solution structure of tear lipocalin: evidence for features that confer promiscuity in ligand binding.

The solution structure of human TL was deduced from the position of the emission peaks after site-directed tryptophan fluorescence (SDTF). The fluorescent amino acid tryptophan was sequentially substituted for each native amino acid in the sequence. Characteristic periodicities for eight beta-strands that comprise the beta-barrel and three alpha-helices were identified. The putative beta-strand I was relatively exposed to solvent, suggesting it does not participate in the formation of the beta-barrel. The beta-strands A and F contain beta-bulges. The average lambda(max) of emission maxima reveals that strand D is at the edge of the barrel and beta-strand H interacts with the main alpha-helical domain. On the basis of the SDTF data, a 3D homology model was constructed for TL and compared to the known crystallographic structures of RBP and beta-lactoglobulin. The small size and splayed open configuration of the E-F hairpin facilitate access of ligands into the cavity mouth of TL as compared to that of RBP with a long overhanging loop that restricts access. In the model of TL, four alanine residues are positioned in the binding site as compared to bulkier residues in the corresponding positions of beta-lactoglobulin. Substitution of A51, A66, A86 to Trp results in a 3-4-fold decrease in binding affinity. The data suggest that the smaller side chains of Ala provide more capacity in the cavity of TL than the bulkier side chains (I56, I71, V92) in the cavity of beta-lactoglobulin. The structural features provide an explanation for the promiscuous binding characteristics exhibited by TL. SDTF provides a general approach for determining the solution structure of many proteins and enhances homology modeling in the absence of high sequence identity.

Functional cavity dimensions of tear lipocalin.

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 beta-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 A) 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 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 inter-sheet distances at the mouth of the crystallized lipocalins ranged from 16-22A. CONCLUSIONS: Tear lipocalin is more promiscuous than beta-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 beta strands.

Endonuclease activity in lipocalins.

Several lipocalins contain conserved amino acid sequences similar to the phosphodiester bond cleavage domain of sugar non-specific magnesium-dependent nucleases of the Serratia marcescens type. His-89 and Glu-127 of the S. marcescens endonuclease are believed to have a role in the active catalytic site by the attack of a water molecule at the phosphorus atom of the bridging phosphate. Tear lipocalin contains both amino acids in analogous regions, and is active as a nuclease. Two forms of beta-lactoglobulin contain only Glu-134 (analogous to Glu-127 of the Serratia nuclease) yet retain nuclease activity equal to or greater than that of tear lipocalin. However, retinol-binding protein lacks both of these motifs and shows no detectable activity. DNA-nicking activity is decreased by 80% in the mutant of tear lipocalin that replaces Glu-128 but is unchanged by mutations of His-84. The endonuclease activity of tear lipocalin is dependent on the bivalent cations Mg(2+) or Mn(2+) but is decreased at high concentrations of NaCl. These findings indicate that some lipocalins have non-specific endonuclease activity similar in characteristics to the Mg(2+)-dependent nucleases and related to the conserved sequence LEDFXR (where 'X' denotes 'any other residue'), in which the glutamic residue seems to be important for activity.

Resolution of ligand positions by site-directed tryptophan fluorescence in tear lipocalin.

The lipocalin superfamily of proteins functions in the binding and transport of a variety of important hydrophobic molecules. Tear lipocalin is a promiscuous lipid binding member of the family and serves as a paradigm to study the molecular determinants of ligand binding. Conserved regions in the lipocalins, 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 strand of holo- and apo-tear lipocalin using spectroscopic methods including circular dichroism analysis and site-directed tryptophan fluorescence. Apo-tear lipocalin shows the same general structural characteristics as holo-tear lipocalin including alternating periodicity of a beta-strand, orientation of amino acid residues 105, 103, 101, and 99 facing the cavity, and progressive 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 associated with blue shifted spectra. The collisional rate constants indicate that these residues are not less exposed to solvent in holo-tear lipocalin than in apo-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 apo-proteins. These findings are consistent with the general hypothesis that the F-G loop in the holo-proteins of the lipocalin family is available for receptor interactions and delivery of ligands to specific targets. Site-directed tryptophan fluorescence was used in combination with a nitroxide spin labeled fatty 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 interact 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 fluorescence with quenching by nitroxide labeled species has broad applicability in probing specific interactions in the solution structure of proteins and provides dynamic information that is not attainable by X-ray crystallography.

Tear lipocalins: potential lipid scavengers for the corneal surface.

PURPOSE: To investigate the dynamic effect of tear lipocalins (TLs), the major lipid-binding protein in tears, at aqueous-cornea and lipid-aqueous interfaces, and their potential contribution to surface tension in the tear film. METHODS: Human apo- and holo-TLs were applied to the aqueous subphase in a Langmuir trough, and changes in surface pressure were measured. Changes in the contact angle of tear components were observed on Teflon and ferric-stearate-treated surfaces. A nitroxide-labeled derivative of lauric acid and a fluorescence-labeled derivative of palmitic acid were used to monitor the dynamic interaction of lipid removed from a hydrophobic surface by the major tear components in solution. RESULTS: TLs increase the surface pressure at the aqueous-air interface by penetrating, spreading, and rearranging on the surface. Apo-TLs show a longer diffusion-dependent induction time than holo-TLs due to more extensive oligomerization of the apoprotein. Kinetic analysis of relaxation time suggests that apo-TLs have more rapid surface penetration and rearrangement than holo-TLs, indicative of a more flexible structure in apo-TLs. TLs reduce the contact angle of solutions on lipid films, a property that is greater with TLs than other tear proteins. TLs, unlike lysozyme and lactoferrin, remove labeled lipids from hydrophobic surfaces and deliver them into solution. CONCLUSIONS: TLs are potent lipid-binding proteins that increase the surface pressure of aqueous solutions while scavenging lipids from hydrophobic surfaces and delivering them to the aqueous phase of tears. These data suggest important functional roles for TLs in maintaining the integrity of the tear film.

Interaction of tear lipocalin with lysozyme and lactoferrin.

The interaction of human tear lipocalin with lysozyme and lactoferrin was studied by electron paramagnetic resonance (EPR) spectroscopy. TL mutants I98C and F99C were spin labeled with MTSL and its derivative. The spectra demonstrated that at sites C98 and C99 the mobility of the nitroxides was reduced in the presence of lysozyme, lactoferrin, but not albumin. The reduced mobility was manifested as a reduction in side chain motion and backbone fluctuations. The overall correlation time of tear lipocalin, measured by MTSL derivative-labeled F99C, was prolonged in the presence of lysozyme and lactoferrin indicating that the interaction involves direct contact. The effect was mitigated at high salt concentration suggesting an electrostatic interaction of the molecules. The reduction in side chain mobility at C98 and C99 of tear lipocalin was observed in tears. Taken together, the data indicate that tear lipocalin interacts with both lysozyme and lactoferrin and suggest that they may function in concert with one another.

Side chain mobility and ligand interactions of the G strand of tear lipocalins by site-directed spin labeling.

Side chain mobility, accessibility, and backbone motion were studied by site-directed spin labeling of sequential cysteine mutants of the G strand in tear lipocalins (TL). A nitroxide scan between residues 98 and 105 revealed the alternating periodicity of mobility and accessibility to NiEDDA and oxygen, characteristic of a beta-strand. Residue 99 was the most inaccessible to NiEDDA and oxygen. EPR spectra with the fast relaxing agent, K(3)Fe(CN)(6), exhibited two nitroxide populations for most residues. The motionally constrained population was relatively less accessible to K(3)Fe(CN)(6) because of dynamic tertiary contact, probably with side chain residues of adjacent strands. With increasing concentrations of sucrose, the spectral contribution of the immobile component was greater, indicating a larger population with tertiary contact. Increased concentrations of sucrose also resulted in a restriction of mobility of spin-labeled fatty acids which were bound within the TL cavity. The data suggest that sucrose enhanced ligand affinity by slowing the backbone motion of the lipocalin. The correlation time of an MTSL derivative (I) attached to F99C resulted in the lack of side chain motion and therefore reflects the overall rotation of the TL complex. The correlation time of F99C in tears (13.5 ns) was the same as that in buffer and indicates that TL exists as a dimer under native conditions. TL-spin-labeled ligand complexes have a shorter correlation time than the protein alone, indicating that the fatty acids are not rigidly anchored in the cavity, but move within the pocket. This segmental motion of the ligand was modulated by protein backbone fluctuations. Accessibility studies with oxygen and NiEDDA were performed to determine the orientation and depth of a series of fatty acid derivatives in the cavity of TL. Fatty acids are oriented with the hydrocarbon tail buried in the cavity and the carboxyl group oriented toward the mouth. In general, the mobility of the nitroxide varied according to position such that nitroxides near the mouth had greater mobility than those located deep in the cavity. Nitroxides positioned up to 16 carbon units from the hydrocarbon tail of the ligand are motionally restricted and inaccessible, indicating the cavity extends to at least this depth. EPR spectra obtained with and without sucrose showed that the intracavitary position of lauric acid in TL is similar to that in beta-lactoglobulin. However, unlike beta-lactoglobulin, TL binds 16-doxyl stearic acid, suggesting less steric hindrance and greater promiscuity for TL.

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

The principal lipid binding protein in tears, tear lipocalin (TL), binds acid and the fluorescent fatty acid analogs, DAUDA and 16-AP at one site TL compete for this binding site. A fluorescent competitive binding assay revealed that apo-TL has a high affinity for phospholipids and stearic acid (Ki) of 1.2 microM and 1.3 microM, respectively, and much less affinity for cholesterol (Ki) of 15.9 of the hydrocarbon chain. TL binds most strongly the least soluble lipids permitting these lipids to exceed their maximum solubility in aqueous solution. These 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 substitution resulted in some loss os secondary structure, relaxation of aromatic side chain rigidity, decreased binding affinity for DAUDA and destabilization of structure. Mutants of TL, W17Y, and W17F showed a higher binding affinity for DAUDA than wild-type TL. Comparison of the results of the tryptophan 17 substitution in lipocalin with those of tryptophan 19 substitution in beta-lactoglobulin revealed important differences in binding characteristics that reflect the functional heterogeneity within the lipocalin family.

Structural changes in human tear lipocalins associated with lipid binding.

Structural and conformational changes in tear lipocalins were detected in association with ligand binding and release. Circular dichroism measurements demonstrated that ligand binding induces beta structure formation, aromatic side chain asymmetry, and a more rigid state in tear lipocalins (TL). The exposure of the tyrosyl component is less in apo-TL than in holo-TL. The sole tryptophan residue, Trp17, is buried in both holo- and apo-TL. The steady state exposure of Trp17 is the same in holo- and apo-TL, but the dynamic exposure is two-fold greater in apo-TL. Maneuvers to unfold the protein with urea or incubation in an acidic environment resulted in increased exposure of aromatic amino acids. Electron paramagnetic resonance studies verified that lipids are liberated from TL in an acidic environment. Acidic pH promotes conformational changes in TL involving aromatic residues, particularly the conserved residue Trp17. These changes are associated with lipid release. The liberation of lipid from the cavity of TL under acidic conditions involves a molten globule state of the protein. We postulate that TL, exposed to the steep surface pH gradient that exists at lipid-aqueous interfaces, would release lipid in association with a molten globule transition. The data suggest a plausible regulatory mechanism for lipid delivery from lipocalins at the tear film surface.

A conserved disulfide motif in human tear lipocalins influences ligand binding.

Structural and functional characteristics of the disulfide motif have been determined for tear lipocalins, members of a novel group of proteins that carry lipids. Amino acid sequences for two of the six isolated isoforms were assigned by a comparison of molecular mass measurements with masses calculated from the cDNA-predicted protein sequence and available N-terminal protein sequence data. A third isoform was tentatively sequence assigned using the same criteria. The most abundant isoform has a measured mass of 17 446.3 Da, consistent with residues 19-176 of the putative precursor (calculated mass 17 445.8 Da). Chemical derivatization of native and reduced/denatured protein confirmed the presence of a single intramolecular disulfide bond in the native protein. Reactivity of native, reduced, and denatured protein with 4-pyridine disulfide and dithiobis(2-nitrobenzoic acid) indicated that access to the free cysteine is markedly restricted by the intact disulfide bridge. Mass measurements of tryptic fragments identified C119 as the free cysteine and showed that the single intramolecular disulfide bond joined residues C79 and C171. Circular dichroism indicated that tear lipocalins have a predominant beta-pleated sheet structure (44%) that is essentially retained after reduction of the disulfide bond. Circular dichroism in the far-UV showed reduced molecular asymmetry and enhanced urea-induced unfolding with disulfide reduction indicative of relaxation of protein structure. Circular dichroism in the near-UV shows that the disulfide bond contributes to the asymmetry of aromatic sites. The effect of disulfide reduction on ligand binding was monitored using the intrinsic optical activity of bound retinol. The intact disulfide bond diminishes the affinity of tear lipocalins for retinol and restricts the displacement of native lipids by retinol. Disulfide reduction is accompanied by a dramatic alteration in ligand-induced conformational changes that involves aromatic residues. The disulfide bridge in tear lipocalins is important in conferring protein rigidity and influencing ligand affinity. The disulfide bond appears highly conserved so that these findings may have implications for the entire lipocalin superfamily.

Solution structure by site directed tryptophan fluorescence in tear lipocalin.

The solution structure of the G strand of human tear lipocalin was deduced by site directed tryptophan fluorescence (SDTF). The fluorescent amino acid, tryptophan, was sequentially substituted for each native amino acid in the sequence of the G strand. The fluorescent properties resolved alternating periodicity as predicted for beta sheet structure, twists in the beta sheet, strand orientation in the lipocalin cavity, and the relative depth of residues in the cavity. A distribution of microstates with various orientations of dipoles in the side chain environments of the G strand revealed mobility on the nanosecond time scale. SDTF is broadly applicable to most proteins and will complement x-ray crystallography, site directed spin labeling by electron paramagnetic resonance (EPR), and nuclear magnetic resonance (NMR) in the determination of solution structure.