Preferential lipid association and mode of penetration of apocytochrome c in mixed model membranes as monitored by tryptophanyl fluorescence quenching using brominated phospholipids

The fluorescence of the single tryptophan residue at position 59 in apocytochrome c, the biosynthetic precursor of the inner mitochondrial membrane protein cytochrome c, was studied in small unilamellar vesicles composed of phosphatidylserine (PS) and phosphatidylcholine (PC) with or without specifically Br-labelled acyl chains at the sn-2 position. The protein has a very high affinity for PS-containing vesicles (dissociation constant Kd less than 1 microM). From the relative quenching efficiency by the brominated phospholipids, it could be concluded that the protein specifically associates with the PS component in mixed vesicles and that maximal quenching occurred with phospholipids in which the bromine was present at the 6,7-position of the 2-acyl chain suggesting that (part of) the bound protein penetrates 7-8 A deep into the hydrophobic core of the bilayer.     

Interaction of antioxidants with depth-dependent fluorescence quenchers and energy transfer probes in lipid bilayers.

Three fluorescent, lipophilic, heterocyclic antioxidants were incorporated into lipid bilayers and exposed to depth-dependent nitroxyl fatty acid quenchers. The Stern-Volmer plots curved upward at low quencher concentrations. Quantitative analysis of the results showed that this behavior is consistent with complex formation between quencher and fluorescent antioxidant, where the complex is 2-3 times more fluorescent than the parent fluorophore. At higher quencher concentrations, both free antioxidant and 'bright complex' are quenched dynamically, albeit quenching of the latter is less efficient. The complex probably results from ionic, hydrogen bond and pi-pi interactions. Formation of such a 'bright complex' is also observable in a homogeneous solution of the reactants in cyclohexane. Additional evidence for the complexation of these antioxidants with fatty acids in lipid bilayers is provided by the fact that energy transfer from the antioxidants to anthroyloxy fatty acids occurs at surface concentrations where radiative energy transfer between free molecules should be not be efficient. For directly probing the relative depths of these fluorophores in lipid bilayers we used the aqueous quenchers acrylamide and iodide. They showed that in terms of increasing depth in the bilayer, the order was U-78, 517f < U-78,518e < U-75,412e. Our results, in toto, demonstrate that the Lazaroid antioxidants are incorporated into the lipid bilayer where they occupy strictly defined positions and orientations. Complexation with fatty acyl chains should be mechanistically relevant, since it may enhance antioxidant activity by hindering free radical chain propagation.     

Fluorescence quenching in model membranes: phospholipid acyl chain distributions around small fluorophores

Fluorescence quenching in lipid bilayers is treated by a new approach based on calculation of the probability distribution of quenching and nonquenching acyl chains around a fluorophore. The effect of acyl lattice site dependence (i.e., correlations of phospholipid sister chain occupancy of neighbor sites) was modeled by use of Monte Carlo simulations of acyl chain occupancy. This explicit accounting of site occupancy correlation was found to fit observed quenching behavior better than did a model wherein phospholipid quenchers are considered to be independent. A key aspect of this approach is to evaluate the rate for quenching in a bilayer composed of pure quenching lipid. In order to evaluate this quenching rate, and also to provide a strong test of the calculated probability distributions, we synthesized lipids with both acyl chains labeled with a quenching moiety (Br or nitroxide), as well as the more usual single-chain quenchers. The fluorescence of tryptophan octyl ester (TOE), and of the 1,6-diphenyl-1,3,5-hexatriene (DPH) derivatives trimethylammonium-DPH (TMA-DPH) and 1-lauroyl-2-(DPH-propionyl)phosphatidylcholine (DPH-PC), was examined. We obtained consistent results with all the fluorophores and quenchers indicating that up to 18 neighboring acyl sites can contribute to quenching, corresponding to two shells of acyl sites on a hexagonal lattice. Calculated discrete distributions of fluorescence intensities were converted into fluorescence lifetimes and compared with Gaussian and Lorentzian continuous lifetime distributions. The fluorescence quenching theory presented here may be used to explain quantitatively the heterogeneity of fluorophore environments in multicomponent membranes.     

Perturbation of phospholipid bilayers by DDT

The localization of the effects of DDT (5–50 mol%) addition on the acyl chain dynamics in unilamellar vesicles of two phosphatidylcholines (DPPC and egg PC) has been investigated by steady-state fluorescence polarization of a series of n-(9-anthroyloxy) fatty acids (n = 2, 6, 9, 12 and 16) whose fluorophore is located at a graded series of depths from the surface to the centre of the bilayer. The results show that DDT is a fluidizer of DPPC and egg PC bilayers. The increase in microviscosity of DPPC bilayers at 23°C begins at the centre of the bilayer (5 mol% DDT) and proceeds outward to the surface with increasing concentration of DDT (17 mol%). This pattern of effects is not evident in fluid bilayers of DPPC at 54°C or egg PC at 23°C. DDT (33 mol%) also lowers the phase transition temperature of DPPC bilayers by approximately 2 Cdeg. DDT (17 mol%) had no effect on the mean excited fluorescence life-time of 2-AP and 12-AS in DPPC, DOPC and egg PC bilayers. No quenching of 2-AP fluorescence was evident.     

Glycosphingolipids: 2H NMR study of the influence of carbohydrate headgroup structure on ceramide acyl chain behavior in glycolipid-phospholipid bilayers

Galactosyl- and glucosylceramide, globoside, and dihydrolactosylceramide, bearing [2,2-2H2]stearic acid, have been studied at a concentration of 10 mol% in bilayers of dimyristoylphosphatidylcholine by 2H NMR. The quadrupolar splitting delta vQ of the C2 deuterons were measured at several temperatures in the range of 30-60 degrees C. Spin-lattice relaxation times T1 of C2 deuterons were determined in the same temperature range for all lipids but globoside. T1 values at 30 and 50 degrees C were unexpectedly short (6-8 ms), indicating reduced mobility of the ceramide acyl chains compared to that of the host phospholipid. At all temperatures, both delta vQ and T1 were essentially identical for the monoglycosylated species, GalCer and GlcCer, indicating that the order and dynamics of the upper portion of the fatty acyl chain are insensitive to this small change in the headgroup structure. In the case of globoside, where the glycolipid headgroup is equivalent to that of GlcCer extended by three sugar residues, values for the quadrupolar splittings associated with the acyl chain C2-position were very close to those obtained for Gal- and GlcCer. In contrast, the delta vQ values obtained for the diglycosyl species, LacCer, were significantly different at all temperatures. This different behavior of LacCer relative to that of the other glycolipids most likely originates from an orientational change of the acyl chain at the C2-position due to the absence of a 4,5 double bond in dihydrosphingosine. T1 values for the GlcCer and GalCer systems increased with temperature, indicating that the motions responsible for relaxation were in the short correlation time regime.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of sphingomyelin acyl chain heterogeneity upon properties of raft-like membranes

Sphingomyelin (SM) is a main component of lipid rafts and characteristic of abundance of long and saturated acyl chains. Recently, we reported that fluorescence-labeled lipids including C16:0 and C18:0SMs retained membrane behaviors of inherent lipids. Here, we newly prepared fluorescent SMs with longer acyl chains, C22:0 and C24:1, for observing their partition and diffusion in SM/cholesterol (chol)/dioleoylphosphatidylcholine (DOPC) bilayers. Although fluorescent C24:1SM underwent a uniform distribution between ordered (Lo) and disordered (Ld) phases, other fluorescent SMs with saturated acyl chains were preferentially distributed in the Lo phase. Interestingly, when the acyl chains of fluorescent and membrane SMs are different, distribution of fluorescent SM to the Lo phase was reduced compared to when the acyl chains are the same. This tendency was also observed for C16:0SM/C22:0SM/chol/DOPC quaternary bilayers, where the minor SM was more excluded out of the Lo phase than the major SM. We also found that the coexistence of SMs induces SM efflux out of the Lo phase and simultaneous DOPC influx to the Lo phase, consequently reducing the difference in fluidity between the two phases. These results suggest that physicochemical properties of lipid rafts are regulated by the acyl chain heterogeneity of SMs.     

Interaction of the alpha-helices of apolipophorin III with the phospholipid acyl chains in discoidal lipoprotein particles: a fluorescence quenching study.

Quenching of tryptophan fluorescence by nitroxide-labeled phospholipids and nitroxide-labeled fatty acids was used to investigate the lipid-binding domains of apolipophorin III. The location of the Trp residues relative to the lipid bilayer was investigated in discoidal lipoprotein particles made with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and five different single-Trp mutants of apoLp-III. A comparison of the quenching efficiencies of phospholipids containing nitroxide groups at the polar head, and at positions 5 and 16 of the sn-2 acyl chain, indicated that the protein is interacting with the acyl chains of the phospholipid along the periphery of the bilayer of the discoidal lipoprotein. N-Bromosuccinimide readily abolished 100% of the fluorescence of all Trp residues in the lipid-bound state. Larger quenching rates were observed for the Trp residues in helices 1, 4, and 5 than for those located in helices 2 and 3, suggesting differences between the interaction of these two groups of helices. However, the extent of Trp fluorescence quenching observed in lipoproteins made with any of the mutants was comparable to that reported for deeply embedded Trp residues, suggesting that all Trp residues interact with the phospholipid acyl chains. This study provides the first experimental evidence of a massive interaction of the alpha-helices of apoLp-III with the phospholipid acyl chains in discoidal lipoproteins. The extent of interaction deduced is consistent with the apolipoprotein adopting a highly extended conformation.     

Effect of cholesterol on molecular order and dynamics in highly polyunsaturated phospholipid bilayers.

The effect of cholesterol on phospholipid acyl chain packing in bilayers consisting of highly unsaturated acyl chains in the liquid crystalline phase was examined for a series of symmetrically and asymmetrically substituted phosphatidylcholines (PCs). The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic structural properties of bilayers containing 30 mol % cholesterol. The bilayers were composed of symmetrically substituted PCs with acyl chains of 14:0, 18:1n9, 20:4n6, or 22:6n3, containing 0, 1, 4, or 6 double bonds, respectively, and mixed-chain PCs with a saturated 16:0 sn-1 chain and 1, 4, or 6 double bonds in the sn-2 chain. DPH excited-state lifetime was fit to a Lorentzian lifetime distribution, the center of which was increased 1-2 ns by 30 mol % cholesterol relative to the cholesterol-free bilayers. Lifetime distributions were dramatically narrowed by the addition of cholesterol in all bilayers except the two consisting of dipolyunsaturated PCs. DPH anisotropy decay was interpreted in terms of the Brownian rotational diffusion model. The effect of cholesterol on both the perpendicular diffusion coefficient D perpendicular and the orientational distribution function f(theta) varied with acyl chain unsaturation. In all bilayers, except the two dipolyunsaturated PCs, 30 mol % cholesterol dramatically slowed DPH rotational motion and restricted DPH orientational freedom. The effect of cholesterol was especially diminished in di-22:6n3 PC, suggesting that this phospholipid may be particularly effective at promoting lateral domains, which are cholesterol-rich and unsaturation-rich, respectively. The results are discussed in terms of a model for lipid packing in membranes containing cholesterol and PCs with highly unsaturated acyl chains.     

Cyclopentanoid analogs of dipalmitoyl phosphatidic acid: effect of backbone geometry on thermotropic properties

Seven geometrical or positional isomers of dipalmitoyl cyclopentanophosphoric acid (DPCPA) have been synthesized and studied: 1,3/2-1P (I); 1,2/3-1P (II); 1,2/3-3P (III), 1,2,3/0-1P (IV); 1,2,3/0-2P (V); 1,3/2-2P (VI); 1,2/3-2P (VII). When dispersed in 0.1 M Tris-HCl at pH 7.4, I-VII gave thermal transitions (Tc) of 60.0 degrees, 59.0 degrees, 56.8 degrees, 55.3 degrees, 38.3 degrees, 36.8 degrees and 34.0 degrees C, respectively, as measured by differential scanning calorimetry (DSC). When the lipids were dispersed at pH 9.5 in 0.1 M borate, Tc of I-IV decreased, whereas Tc of V-VII increased. In contrast, at pH 1.5 in 0.1 M HCl/KCl, Tc of I-IV decreased slightly, but Tc of V-VII rose markedly. To determine the effect of head group geometry and substitution pattern on acyl chain motion, EPR spectra of 1-palmitoyl, 2-[16-doxylstearoyl]-glycero-3-phosphoric acid in bilayers of DPCPA isomers were acquired. Abrupt spectral changes occurred at temperatures closely correlating with transition temperatures observed by DSC. These results have led to the conclusions that: (i) isomers I-IV containing vicinal acyl chains form bilayers that exhibit structural transitions at temperatures higher than those at which transitions are exhibited by isomers V-VII which have a polar phosphate group interposed between the two chains; (ii) the effects of differences in backbone structure are transmitted down the entire length of the acyl chains; (iii) the orientation of the cyclopentane ring in the isomers I-IV is significantly different from that in isomers V-VII at pH values where the phosphate group is doubly negatively charged.     

Effects of cholesterol on conformational disorder in dipalmitoylphosphatidylcholine bilayers. A quantitative IR study of the depth dependence

A method originally proposed by Snyder and Poore [(1973) Macromolecules 6, 708-715] as a specific probe of trans-gauche isomerization in hydrocarbon chains and recently applied [Mendelsohn et al. (1989) Biochemistry 28, 8934-8939] to the quantitative determination of phospholipid acyl chain conformational order is utilized to monitor the effects of cholesterol at various depths in dipalmitoylphosphatidylcholine (DPPC) bilayers. The method is based on the observation that the CD2 rocking modes from the acyl chains of specifically deuterated phospholipids occur at frequencies in the Fourier transform infrared spectrum which depend upon the local geometry (trans or gauche) of the C-C-C skeleton surrounding a central CD2 group. Three specifically deuterated derivatives of DPPC, namely, 4,4,4',4'-d4 DPPC (4-d4 DPPC), 6,6,6',6'-d4 DPPC (6-d4 DPPC), and 12,12,12',12'-d4 DPPC (12-d4 DPPC), have been synthesized, and the effects of cholesterol addition at 2:1 DPPC/cholesterol (mol:mol) on acyl chain order at various temperatures have been determined. At 48 degrees C, cholesterol inhibits gauche rotamer formation by factors of approximately 9 and approximately 6 at positions 6 and 4, respectively, of the acyl chains, thus demonstrating a strong ordering effect in regions of the bilayer where the sterol rings are presumed to insert parallel to the DPPC acyl chains. In contrast, the ability of the sterol to order the acyl chains is much reduced at the 12-position. The sterol demonstrates only a slight disordering of phospholipid gel phases. Finally, the contributions of different classes of gauche conformers to the spectra have been determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

A variety of novel lipid A structures obtained from Francisella tularensis live vaccine strain

F. tularensis is a Gram-negative coccobacillus that causes tularemia. Its LPS has nominal biological activity. Currently, there is controversy regarding the structure of the lipid A obtained from F. tularensis live vaccine strain (LVS). Therefore, to resolve this controversy, the purification and structural identification of this LPS was crucial. To achieve this, LPS from F. tularensis LVS was acid hydrolyzed to obtain crude lipid A that was methylated and purified by HPLC and the fractions were analyzed by MALDI-TOF MS. The structure of the major lipid A species was composed of a glucosamine disaccharide backbone substituted with four fatty acyl groups and a phosphate (1-position) with a molecular mass of 1505. The major lipid A component contained 18:0[3-O(16:0)] in the distal subunit and two 18:0(3-OH) fatty acyl chains at the 2- or 3-positions of the reducing subunit. Additional variations in the lipid A species include: heterogeneity in fatty acyl groups, a phosphate or a phosphoryl galactosamine at the 1-position, and a hexose at the 4' or 6' position, some of which have not been previously described for F. tularensis LVS. This analysis revealed that lipid A from F. tularensis LVS is far more complex than originally believed.     

Fluorescence quenching in lecithin and lecithin/cholesterol liposomes by parmagenetic lipid analogues. Introduction of a new probe approach.

1. Perylene, whether incorporated into lecithin or lecithin/cholesterol (1:1) liposomes, exhibits identical fluorescence spectra, but fluorescence in the presence of cholesterol is enhanced by 30-50%. 2. The fluorescence of perylene in pure dipalmitoyllecithin vesicles increases sharply at the transition temperature (Tt equals 41 degrees C). No such fluorescence jump is observed in lecithin/cholesterol (1:1) micelles. 3. In lecithin liposomes maximal quenching of perylene fluorescence at 25 degrees C is effected by cholestane spin label (80%) followed by androstane spin label (70%), 5-nitroxide stearate (60%) and 16-nitroxide stearate (50%). 4. In liposomes containing 5 mol % cholesterol these differences are reduced; however, the sequence of quenching efficiencies is the same except for the nitroxide stearates, which interchange their positions. 5. 5. Paramagnetic quenching of perylene fluorescence is stable below 35 degrees C and above 45 degrees C, but decreases sharply about the phase-transition temperature of dipalmitoyllecithin. 6. In lecithin/cholesterol (1:1, molar ratio) lipsomes fluorescence quenching diminishes linearly, but only slightly, with increasing temperature. 7. Cholestane spin label and androstane spin label at concentrations of greater than 20 mol % themselves suppress the quenching discontinuity at Tt, indicating a cholesterol-like structural effect. 8. The quenching phenomena observed are attributed to a non-random accommodation of fluorophore and quencher molecules (co-clustering) below the phase transition and a statistical distribution of both impurities above Tt. 9. In the presence of cholesterol the clustering tendencies are reduced or even eliminated; this is compatible with the concept that cholesterol fluidizes the phosphatide acyl chains below the transtion temperature.     

Orientation of LamB signal peptides in bilayers: influence of lipid probes on peptide binding and interpretation of fluorescence quenching data.

The orientation in lipid bilayers of the signal sequence of the bacterial protein LamB was studied using binding, circular dichroism, and fluorescence quenching experiments. Measurements were made of binding modifications caused by the incorporation of lipid probes (brominated or nitroxide-labeled phospholipids) used in the parallax fluorescence quenching method of determining peptide penetration depth [Abrams, F. S., and London, E. (1992) Biochemistry 31, 5312-5322]. The signal peptide bound to a similar extent to neutral bilayers composed of either egg phosphatidylcholine (EPC) or phosphatidylcholines brominated at various positions on their acyl chains. The fluorescence of a tryptophan in either the 18 or 24 position of the peptide was quenched more by bromines in the 6 and 7 than in the 9 and 10 positions on the lipid hydrocarbon chain. Parallax calculations showed that tryptophan-18 was located only 4 A from the hydrocarbon-water interface, consistent with the peptide adopting a "hammock" configuration in the bilayer, with both termini exposed to the aqueous phase and the central alpha-helix located near the hydrocarbon-water interface. In contrast, the incorporation of 10% nitroxide-labeled lipids into EPC bilayers modified peptide binding in a manner dependent on the position of the nitroxide on the hydrocarbon chain; 7-Doxyl PC reduced the percent peptide bound by about one-half, whereas 12-Doxyl PC had little effect on binding. These binding differences modified tryptophan quenching by these probes, making parallax analysis problematical. In the presence of the positively charged LamB peptide, the incorporation of negatively charged phospholipids into EPC bilayers increased the level of peptide binding and modified tryptophan quenching by nitroxide probes. These results suggest that the nitroxide probe could be partially excluded from negatively charged lipid domains where the peptide preferentially bound. Quite different binding and quenching results were obtained with a negatively charged peptide analogue, showing that the charge on both the peptide and bilayer affects peptide-nitroxide probe interactions.     

The membrane interaction of amphiphilic model peptides affects phosphatidylserine headgroup and acyl chain order and dynamics. Application of the "phospholipid headgroup electrometer" concept to phosphatidylserine

Deuterium nuclear magnetic resonance (2H NMR) was used to study the interaction of amphiphilic model peptides with model membranes consisting of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine deuterated either at the beta-position of the serine moiety ([2-2H]DOPS) or at the 11-position of the acyl chains ([11,11-2H2]DOPS). The peptides are derived from the sequences H-Ala-Met-Leu-Trp-Ala-OH (AX, one-letter code with X = MLWA) and H-Arg-Met-Leu-Trp-Ala-OH (RX+) and contain a positive charge of +1 (AXme+) or +2 (RXme2+) at the amino terminus or one positive charge at each end of the molecule (AXetN2+). Upon titration of dispersions of DOPS with the peptides, the divalent peptides show a similar extent of binding to the DOPS bilayers, which is larger than that of the single charged peptide. Under these conditions the values of the quadrupolar splitting (delta vq) of both [2-2H]DOPS and [11,11-2H2]DOPS are decreased, indicating that the peptides reduce the order of both the DOPS headgroup and the acyl chains. The extent of the decrease depends on the amount of peptide bound and on the position of the charged moieties in the peptide molecule. The effects exerted by the peptides on the delta vq value of [2-2H]DOPS are consistent with the PS headgroup responding as a molecular electrometer to the surface charge resulting from the presence of the peptides in the lipid-water interface. The effects on the acyl chain deuterons are in agreement with a localization of the peptides intercalated in between the lipid headgrouops.(ABSTRACT TRUNCATED AT 250 WORDS)     

Turnover of fatty acids in the 1-position of phosphatidylethanolamine in Escherichia coli

Phosphatidylethanolamine is the major membrane phospholipid of Escherichia coli, and two experimental approaches were used to investigate the metabolic activity of the fatty acids occupying the 1-position of this phospholipid. [3H]Acetate pulse-chase experiments with logarithmically growing cells indicated that 3-5% of the acyl groups were removed from the phosphatidylethanolamine pool/generation. The reacylation aspect of the turnover cycle was demonstrated by the incorporation of fatty acids into the 1-position of pre-existing phosphatidylethanolamine when de novo phospholipid biosynthesis was inhibited using the plsB acyltransferase mutant. 2- Acylglycerophosphoethanolamine would be the intermediate in a 1-position turnover cycle, and this lysophospholipid was identified as a membrane component that could re-esterified by a membrane-bound acyltransferase. The acyltransferase either utilized acyl-acyl carrier protein directly as an acyl donor or activated fatty acids for acyl transfer in the presence of ATP and Mg2+. Acyl-acyl carrier protein was also indicated as an intermediate in the latter reacylation reaction by the complete inhibition of phosphatidylethanolamine formation from fatty acids by acyl carrier protein-specific antibodies and by the observation that the inhibition of the acyltransferase by LiCl was reversed by the addition of acyl carrier protein. Coenzyme A thioesters were not substrates for this acyltransferase. These results suggest the existence of a metabolic cycle for the utilization of 1-position acyl moieties of phosphatidylethanolamine followed by the resynthesis of this membrane phospholipid from 2- acylglycerophosphoethanolamine by an acyl carrier protein-dependent 1-position acyltransferase.     

A differential scanning calorimetry study of phosphocholines mixed with paclitaxel and its bromoacylated taxanes

High sensitivity differential scanning calorimetry (DSC) was used to investigate the thermotropic phase properties of binary mixtures of disaturated phosphocholines (PCs) and alpha-bromoacyl taxane derivatives. The alpha-bromoacyl taxanes were synthesized as hydrolyzable hydrophobic prodrugs of paclitaxel. The PCs used were 1, 2-dimyristoyl-sn-glycero-3-phosphatidyl-choline (DMPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1, 2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). The bromoacyl chain lengths of the taxane prodrugs were varied from 6 to 12 or 16 carbons. For comparison, paclitaxel and PC mixtures were also examined. DSC data from DPPC and bromoacyl taxane mixtures showed a complete abolition of the pretransition and significant broadening of the main phase transition with increasing amounts of bromoacyl taxane prodrugs. The effects were more pronounced with the long-chain compared to the short-chain prodrugs. Under equivalent DSC conditions, the short-chain DMPC showed greater changes in thermotropic phase behavior than with DPPC on taxane addition, suggesting an enhanced degree of association with the fluid-type bilayers. Under similar conditions, the long-chain DSPC bilayers showed a far less significant change in phase behavior on taxane addition than DPPC. These changes were also chain length-dependent for both the PCs and the taxane prodrugs. In contrast, PC and paclitaxel (lacking the acyl chain) mixtures under similar conditions showed insignificant changes in the endotherms, suggesting only slight insertion of the molecule into the PC bilayers. From the DSC data it is apparent that taxane prodrugs solvated in DMPC bilayers more than in DPPC and DSPC bilayers, and taxane prodrugs with longer acyl chains were able to associate with PCs better than those with shorter chain prodrugs. DSC data also suggest that paclitaxel was poorly associated with any of the PCs. In general, the amount of taxane association with bilayers decreased in order: DMPC > DPPC > DSPC. In contrast, the transition enthalpy (DeltaH) of DMPC, DPPC, and DSPC mixtures with paclitaxel showed significantly lower enthalpies than with taxane prodrugs. Taken together, the DSC data suggest that the acyl chains of paclitaxel prodrugs have some access into the bilayers via alignment with the acyl chain of the PC component.     

Anisotropic 2H NMR spin-lattice relaxation in L alpha-phase cerebroside bilayers

A series of 2H NMR inversion recovery experiments in the L alpha phase of the cerebroside N-palmitoylgalactosylsphingosine (NPGS) have been performed. In these liquid crystalline lipid bilayers we have observed substantial anisotropy in the spin-lattice relaxation of the CD2 groups in the acyl chains. The form and magnitude of the anisotropy varies with position in the chain, being positive in the upper region, decreasing to zero at the 4-position, and reversing sign at the lower chain positions. It is also shown that addition of cholesterol to the bilayer results in profound changes in the anisotropy. These observations are accounted for by a simple motional model of discrete hops among nine sites, which result from the coupling of two modes of motion--long-axis rotational diffusion and gauche-trans isomerization. This model is employed in quantitative simulations of the spectral line shapes and permits determination of site populations and motional rates. These results, plus preliminary results in sphingomyelin and lecithin bilayers, illustrate the utility of T1 anisotropy measurements as a probe of dynamics in L alpha-phase bilayers.     

Acyl and alk-1-enyl group compositions of the alk-1'-enyl acyl and the diacyl glycerophosphoryl ethanolamines of mouse and ox brain

The ethanolamine phosphoglycerides were prepared from lipid extracts of ox and mouse brains by preparative thin-layer chromatography. The cyclic acetal derivatives of the alk-1-enyl groups were made by treating the ethanolamine phosphoglycerides with 1,3-propanediol. The resulting monoacyl glycerophosphoryl ethanolamines were separated from the unchanged ethanolamine phosphoglycerides by preparative thin-layer chromatography. Methyl ester derivatives of the acyl groups from both of these fractions were prepared by alkaline methanolysis. The cyclic acetal and methyl ester derivatives were analyzed by gas-liquid chromatography. Substantial differences were found in the composition of the side chains when the combined alk-1-enyl and acyl side chains of the alk-1'-enyl acyl glycerophosphoryl ethanolamines were compared with the side chains of the diacyl glycerophosphoryl ethanolamines. The side chains from the 1-position of these two ethanolamine phosphoglycerides are different in chain length and unsaturation as well as in chemical bonding. The acyl groups from the 2-position of the alk-1'-enyl acyl glycerophosphoryl ethanolamines were predominantly unsaturated. Therefore, acyl group compositions of the total ethanolamine phosphoglyceride from brain are of limited value and individual types should be analyzed.     

Lipid hydration and mobility: an interplay between fluorescence solvent relaxation experiments and molecular dynamics simulations

Fluorescence solvent relaxation experiments are based on the characterization of time-dependent shifts in the fluorescence emission of a chromophore, yielding polarity and viscosity information about the chromophore’s immediate environment. A chromophore applied to a phospholipid bilayer at a well-defined location (with respect to the z-axis of the bilayer) allows monitoring of the hydration and mobility of the probed segment of the lipid molecules. Specifically, time-resolved fluorescence experiments, fluorescence quenching data and molecular dynamic (MD) simulations show that 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) probes the hydration and mobility of the sn-1 acyl groups in a phosphatidylcholine bilayer. The time-dependent fluorescence shift (TDFS) of Laurdan provides information on headgroup compression and expansion induced by the addition of different amounts of cationic lipids to phosphatidylcholine bilayers. Those changes were predicted by previous MD simulations. Addition of truncated oxidized phospholipids leads to increased mobility and hydration at the sn-1 acyl level. This experimental finding can be explained by MD simulations, which indicate that the truncated chains of the oxidized lipid molecules are looping back into aqueous phase, hence creating voids below the glycerol level. Fluorescence solvent relaxation experiments are also useful in understanding salt effects on the structure and dynamics of lipid bilayers. For example, such experiments demonstrate that large anions increase hydration and mobility at the sn-1 acyl level of phosphatidylcholine bilayers, an observation which could not be explained by standard MD simulations. If polarizability is introduced into the applied force field, however, MD simulations show that big soft polarizable anions are able to interact with the hydrophilic/hydrophobic interface of the lipid bilayer, penetrating to the level probed by Laurdan, and that they expand and destabilize the bilayer making it more hydrated and mobile.