Cubic phases in hydrated 1:1 and 1:2 dipalmitoylphosphatidylcholine-dipalmitoylglycerol mixtures.

The structures of fully hydrated 1:1 and 1:2 (mol/mol) dipalmitoylphosphatidylcholine (DPPC)-dipalmitoylglycerol (DPG) mixtures were studied by means of small-angle x-ray diffraction. The x-ray diffraction pattern of the 1:1 (mol/mol) DPPC-DPG mixture at 65 degrees C contains three reflections with spacings in the ratio of 1:1/ square root of 2:1/ square root of 3 in addition to reflections of an inverted hexagonal (H11) phase. A possible interpretation of this result is that a cubic phase of the body-centered space group lm3m, with a lattice constant of 23.1 +/- 0.6 nm, is formed. This cubic phase appears at intermediate temperatures between the lamellar and the H11 phases. The 1:2 (mol/mol) DPPC-DPG mixture gives an x-ray diffraction pattern at temperatures higher than the lamellar-to-H11 transition containing a number of reflections that index a cubic phase structure. The space group of the cubic phase was assigned a face-centered group Fd3m with a lattice constant of 16.3 +/- 0.1 nm at 82 degrees C. The possible role of cubic phases in membrane phenomena such as transmembrane signal transduction and fusion is discussed.     

Accelerated formation of cubic phases in phosphatidylethanolamine dispersions.

By means of x-ray diffraction we show that several sodium salts and the disaccharides sucrose and trehalose strongly accelerate the formation of cubic phases in phosphatidylethanolamine (PE) dispersions upon temperature cycling through the lamellar liquid crystalline-inverted hexagonal (Lalpha-HII) phase transition. Ethylene glycol does not have such an effect. The degree of acceleration increases with the solute concentration. Such an acceleration has been observed for dielaidoyl PE (DEPE), dihexadecyl PE, and dipalmitoyl PE. It was investigated in detail for DEPE dispersions. For DEPE (10 wt% of lipid) aqueous dispersions at 1 M solute concentration, 10-50 temperature cycles typically result in complete conversion of the Lalpha phase into cubic phase. Most efficient is temperature cycling executed by laser flash T-jumps. In that case the conversion completes within 10-15 cycles. However, the cubic phases produced by laser T-jumps are less ordered in comparison to the rather regular cubic structures produced by linear, uniform temperature cycling at 10 degrees C/min. Temperature cycles at scan rates of 1-3 degrees C/min also induce the rapid formation of cubic phases. All solutes used induce the formation of Im3m (Q229) cubic phase in 10 wt% DEPE dispersions. The initial Im3m phases appearing during the first temperature cycles have larger lattice parameters that relax to smaller values with continuation of the cycling after the disappearance of the Lalpha phase. A cooperative Im3m --> Pn3m transition takes place at approximately 85 degrees C and transforms the Im3m phase into a mixture of coexisting Pn3m (Q224) and Im3m phases. The Im3m/Pn3m lattice parameter ratio is 1. 28, as could be expected from a representation of the Im3m and Pn3m phases with the primitive and diamond infinite periodic minimal surfaces, respectively. At higher DEPE contents ( approximately 30 wt%), cubic phase formation is hindered after 20-30 temperature cycles. The conversion does not go through, but reaches a stage with coexisting Ia3d (Q230) and Lalpha phases. Upon heating, the Ia3d phase cooperatively transforms into a mixture of, presumably, Im3m and Pn3m phases at about the temperature of the Lalpha-HII transition. This transformation is readily reversible with the temperature. The lattice parameters of the DEPE cubic phases are temperature-insensitive in the Lalpha temperature range and decrease with the temperature in the range of the HII phase.     

Observation of inverted cubic phase in hydrated dioleoylphosphatidylethanolamine membranes

We report the observation of an inverted cubic phase in aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) by small-angle X-ray diffraction. DOPE is a paradigm in the study of nonlamellar phases in biological systems: it exhibits a well-known phase transition from the lamellar (L alpha) to the inverted hexagonal phase (HII) as the temperature is raised. The transition is observed to occur rapidly when a DOPE dispersion is heated from 2 degrees C, where the L alpha phase is stable, to 15 degrees C, where the HII phase is stable. We report on the induction of a crystallographically well-defined cubic lattice that is slowly formed when the lipid dispersion is rapidly cycled between -5 and 15 degrees C hundreds of times. Once formed, the cubic lattice is stable at 4 degrees C for several weeks and exhibits the same remarkable metastability that characterizes other cubic phases in lipid-water systems. X-ray diffraction indicates that the cubic lattice is most consistent with either the Pn3m or Pn3 space group. Tests of lipid purity after induction of the cubic indicate the lipid is at least 98% pure. The cubic lattice can be destroyed and the system reset by cycling the specimen several times between -30 and 2 degrees C. The kinetics of the formation of the cubic are dependent on the thermal history of the sample, overall water concentration, and the extreme temperatures of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

31P NMR and X-ray diffraction study of the effect of photopolymerization on lipid polymorphism.

It was recently shown that oligolamellar vesicles of 3:1 mixtures of dioleoylphosphatidylethanolamine (DOPE) and the photopolymerizable lipid 1,2-bis[10-(2',4'-hexadienoyloxy)decanoyl]-sn-glycero-3-phosphocho line (SorbPC) are destabilized by polymerization of the SorbPC [Lamparski, H., Liman, U., Frankel, D.A., Barry, J.A., Ramaswami, V., Brown, M.F., & O'Brien, D.F. (1992) Biochemistry 31, 685-694]. The current work describes the polymorphic phase behavior of these mixtures in extended bilayers, as studied by 31P NMR spectroscopy and X-ray diffraction. In the NMR experiments, samples with varying degrees of polymerization were slowly raised in temperature, with spectra acquired every 2.5-10 degrees C. In the unpolymerized mixiture, and in those photopolymerized samples where the monomeric SorbPC was decreased by 33% and 51%, an isotropic signal grew progressively until no signal from the lamellar liquid-crystalline (L alpha) phase remained. In the highly polymerized sample with a 90% loss of monomeric SorbPC, less than 20% of the lipids underwent this transition. In none of the samples was an inverted hexagonal phase (HII) observed, under conditions of slow heating to almost 100 degrees C. The X-ray diffraction studies indicated that samples which exhibit the isotropic NMR signal corresponded to a structure exhibiting no well-defined crystalline order, which upon thermal cycling became an inverted cubic phase belonging to either the Pn3m or Pn3 space groups. The temperature of the transition to the cubic precursor decreased as the extent of polymerization increased, demonstrating that photopolymerization of these lipid bilayers can significantly alter the composition and thermotropic phase behavior of the mixture.     

Temperature- and pressure-dependent phase behavior of monoacylglycerides monoolein and monoelaidin.

We used x-ray and neutron diffraction to study the temperature- and pressure-dependent structure and phase behavior of the monoacylglycerides 1-monoelaidin (ME) and 1-monoolein (MO) in excess water. The monoacylglycerides were chosen for investigation of their phase behavior because they exhibit mesomorphic phases with one-, two-, and three-dimensional periodicity, such as lamellar, an inverted hexagonal and bicontinuous cubic phases, in a rather easily accessible temperature and pressure range. We studied the structure, stability, and transformations of the different phases over a wide temperature and pressure range, explored the epitaxial relations that exist between different phases, and established a relationship between the chemical structure of the lipid molecules and their phase behavior. For both systems, a temperature-pressure phase diagram has been determined in the temperature range from 0 to 100 degrees C at pressures from ambient up to 1400 bar, and drastic differences in phase behavior are found for the two systems. In MO-water dispersions, the cubic phase Pn3m extends over a large phase field in the T,p-plane. At temperatures above 95 degrees C, the inverted hexagonal phase is found. In the lower temperature region, a crystalline lamellar phase is induced at higher pressures. The phases found in ME-water include the lamellar crystalline Lc phase, the L beta gel phase, the L alpha liquid-crystalline phase, and two cubic phases belonging to the crystallographic space groups Im3m and Pn3m. In addition, the existence of metastable phases has been exploited. Between coexisting metastable cubic structures, a metric relationship has been found that is predicted theoretically on the basis of the curvature elastic energy approximation only.     

Stable cubic phases in codispersions of glucocerebroside and palmitoyloleoylphosphatidylethanolamine

The effect of glucocerebroside (GlcCer) on the structure and thermotropic phase behavior of aqueous dispersions of palmitoyloleoylphosphatidylethanolamine (POPE) has been examined using simultaneous small-angle and wide-angle X-ray diffraction methods. Binary mixtures of GlcCer:POPE in molar ratios of 2:100, 5:100, 10:100, 20:100, 30:100, and 40:100 were examined in the temperature range 20-90 degrees C. Cubic phase has been observed in binary mixtures comprised of molar ratios greater than 5:100 in the temperature range of 60-90 degrees C upon heating at a rate of 2 degrees C/min. The cubic phase is relatively stable and coexists with inverted hexagonal or lamellar phases. It persists in the codispersions throughout subsequent cooling scans to 30 degrees C. The space group of the cubic phase is determined to be Pn3m or Pn3. The lattice constant of the Pn3m cubic phase was found to be almost constant when it coexists with lamellar liquid-crystal phase. Marked temperature-dependent changes were observed when cubic phase coexists with hexagonal phase or lamellar-gel phases. This is the first report of cubic phases formed by codispersions of glycosphingolipids and phospholipids. The mechanism of cubic phase formation and the interaction between GlcCer and POPE is discussed in terms of the putative biological functions of glycolipids.     

Inverted hexagonal and cubic phases induced by alpha-tocopherol in fully hydrated dispersions of dilauroylphosphatidylethanolamine

The effect of alpha-tocopherol on the thermotropic phase behaviour and structure of aqueous dispersions of 1,2-di-lauryl-sn-glycero-3-phosphoethanolamine was examined by synchrotron X-ray diffraction. The pure phospholipid exhibited a lamellar gel to liquid-crystal phase transition at 30 degrees C on heating at 3 degrees C min(-1) between 10 degrees C and 90 degrees C. The transition was reversible with a temperature hysteresis of 0.3 degrees C on cooling. At temperatures less than 10 degrees C only lamellar gel phase of the pure phospholipid was seen in co-dispersions of up to 20 mol % alpha-tocopherol. The presence of 2.5 mol % alpha-tocopherol caused the appearance of inverted hexagonal phase at temperatures just below the main phase transition temperature that co-existed with the lamellar gel phase. The intensity of scattering from the hexagonal-II phase increased with increasing proportion of alpha-tocopherol in the mixture and in proportions greater than 10 mol % it persisted at temperatures above the main transition and co-existed with the lamellar liquid-crystal phase of the pure phospholipid. At higher temperatures all co-dispersions containing up to 15 mol % alpha-tocopherol showed the presence of cubic phases. These phases indexed a Pn3m or Pn3 space grouping. When the proportion of alpha-tocopherol was increased to 20 mol % the only non-lamellar phase observed was inverted hexagonal phase. This phase co-existed with lamellar gel and liquid-crystal phases of the pure phospholipid, but was the only phase present at temperatures >60 degrees C. The X-ray diffraction data were used to construct a partial phase diagram of the lipid mixture in excess water between 10 degrees and 90 degrees C and up to 20 mol % alpha-tocopherol in phospholipid.     

Interaction of the peptide antibiotic alamethicin with bilayer- and non-bilayer-forming lipids: influence of increasing alamethicin concentration on the lipids supramolecular structures

Incorporation of the helical antimicrobial peptide alamethicin from aqueous phase into hydrated phases of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC) was investigated within a range of peptide concentrations and temperatures by time-resolved synchrotron X-ray diffraction. It was found that alamethicin influences the organizations of the non-bilayer-forming (DOPE) and the bilayer-forming (DOPC) lipids in different ways. In DOPC, only the bilayer thickness was affected, while in DOPE new phases were induced. At low peptide concentrations (<1.10(-4) M), an inverted hexagonal (H(II)) phase was observed as with DOPE dispersions in pure buffer solution. A coexistence of two cubic structures was found at the critical peptide concentration for induction of new lipid/peptide phases. The first one Q224 (space group Pn3m) was identified within the entire temperature region studied (from 1 to 45 degrees C) and was found in coexistence with H(II)-phase domains. The second lipid/peptide cubic structure was present only at temperatures below 16 degrees C and its X-ray reflections were better fitted by a Q212 (P4(3)32) space group, rather than by the expected Q229 (Im3m) space group. At alamethicin concentrations of 1 mM and higher, a nonlamellar phase transition from a Q224 cubic phase into an H(II) phase was observed. Within the investigated range of peptide concentrations, lamellar structures of two different bilayer periods were established with the bilayer-forming lipid DOPC. They correspond to lipid domains of associated and nonassociated helical peptide. The obtained X-ray results suggest that the amphiphilic alamethicin molecules adsorb from the aqueous phase at the lipid head group/water interface of the DOPE and DOPC membranes. At sufficiently high (>1.10(-4) M) solution concentrations, the peptide is probably accommodated in the head group region of the lipids thus inducing structural features of mixed lipid/peptide phases.     

Supramolecular structure of lipopolysaccharide and free lipid A under physiological conditions as determined by synchrotron small-angle X-ray diffraction

Lipopolysaccharides, the major amphiphilic components of the outer leaflet of the outer membrane of Gram-negative bacteria, may assume various three-dimensional supramolecular structures depending on molecular properties (e.g. chemical structure) and on ambient conditions (e.g. temperature, concentration of divalent cations). We applied synchrotron small-angle X-ray diffraction to investigate the supramolecular structures of natural and synthetic Escherichia-coli-type lipid A, of lipid A from Salmonella minnesota, and of rough mutant lipopolysaccharides of E. coli and S. minnesota under physiological water content (greater than 90%) at different temperatures (20, 37, and 55 degrees C) and at different lipid/divalent cation molar ratios (20:1 to 1:1). We found that in the absence of divalent cations rough mutant lipopolysaccharide and free lipid A form unilamellar structures with the main reflections centered around 4.50 nm for free lipid A, 4.80 nm for Re lipopolysaccharide, and 5.90 nm for Rd1 lipopolysaccharide at 20 degrees C, i.e. below the beta----alpha acyl-chain-melting transition temperature. Above this temperature, the reflections are shifted to 4.30 nm for free lipid A (at 55 degrees C), 4.60 nm for Re lipopolysaccharide (at 37 degrees C), and to 5.50 nm for Rd1 lipopolysaccharide (at 37 degrees C). The addition of divalent cations leads (at lower concentrations, i.e. lipid/cation molar ratios 20:1 to 5:1) to sharper reflections expressing a higher state of order and to a shift of the center of the main reflections lying now at 5.10 nm for free lipid A, 6.40 nm for Re and 7.20 nm for Rd1 lipopolysaccharide at 20 degrees C. At higher concentrations of divalent cations (e.g. lipid/cation molar ratio 1:1), an increasing tendency to form nonlamellar, inverted cubic structures is observed which is indicated by the occurrence of another main periodicity and/or of reflections with spacing ratios 1: square root of 2, 1: square root of 3 of the main periodicity. The tendency to assume inverted cubic structures is only weakly pronounced for rough mutant lipopolysaccharides but dominant for free lipid A even at physiological temperature and divalent cation concentration.     

The temperature-composition phase diagram and mesophase structure characterization of monopentadecenoin in water.

The temperature-composition phase diagram of monopentadecenoin, a monoacylglycerol with a cis monounsaturated fatty acid 15 carbon atoms long (C15:1c10) in water was constructed using x-ray diffraction. Low- and wide-angle diffraction patterns were collected from samples of fixed hydration as a function of temperature in the heating direction on x-ray-sensitive film. The temperature and hydration ranges investigated were 0-104 degrees C and 0-60% (w/w) water, respectively. The phases identified in the system include the lamellar crystalline phase, the lamellar liquid crystalline phase, the fluid isotropic phase, and two inverted cubic phases belonging to space groups la3d (Q230) and Pn3m (Q244). Particular attention has been devoted to the issues of phase equilibrium, phase boundary verification, and structure characterization. The phase diagrams of monopentadecenoin, monomyristolein (C14:1c9), and monoolein (C18:1c9) are compared, and the impact of molecular structure on mesophase stability and structure is discussed.     

The effect of peptide/lipid hydrophobic mismatch on the phase behavior of model membranes mimicking the lipid composition in Escherichia coli membranes

The effect of hydrophobic peptides on the lipid phase behavior of an aqueous dispersion of dioleoylphosphatidylethanolamine and dioleoylphosphatidylglycerol (7:3 molar ratio) was studied by (31)P NMR spectroscopy. The peptides (WALPn peptides, where n is the total number of amino acid residues) are designed as models for transmembrane parts of integral membrane proteins and consist of a hydrophobic sequence of alternating leucines and alanines, of variable length, that is flanked on both ends by tryptophans. The pure lipid dispersion was shown to undergo a lamellar-to-isotropic phase transition at approximately 60 degrees C. Small-angle x-ray scattering showed that at a lower water content a cubic phase belonging to the space group Pn3m is formed, suggesting also that the isotropic phase in the lipid dispersion represents a cubic liquid crystalline phase. It was found that the WALP peptides very efficiently promote formation of nonlamellar phases in this lipid system. At a peptide-to-lipid (P/L) molar ratio of 1:1000, the shortest peptide used, WALP16, lowered the lamellar-to-isotropic phase transition by approximately 15 degrees C. This effect was less for longer peptides. For all of the WALP peptides used, an increase in peptide concentration led to a further lowering of the phase transition temperature. At the highest P/L ratio (1:25) studied, WALP16 induced a reversed hexagonal liquid crystalline (H(II)) phase, while the longer peptides still promoted the formation of an isotropic phase. Peptides with a hydrophobic length larger than the bilayer thickness were found to be unable to inhibit formation of the isotropic phase. The results are discussed in terms of mismatch between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer and its consequences for lipid-protein interactions in membranes.     

Formation of monolayers and bilayer foam films from lamellar,inverted hexagonal and cubic lipid phases

This study revealed large distinctions between the lamellar and non-lamellar liquid crystalline lipid phases in their spreading at the air/water interface and propensity to form bilayer foam films. Comparative measurements were made for the lamellar L(alpha), the inverted hexagonal H(II) and the bicontinuous cubic Pn3m phases of the phospholipid dipalmitoleoylphosphatidylethanolamine (DPoPE). With regard to monolayer formation, followed as the decrease of surface tension with time, the best spreading (lowest surface tension) was observed for the L(alpha) phase, and poorest spreading (highest surface tension) was recorded for the H(II) phase. The cubic Pn3m phase of DPoPE, induced by temperature cycling, retained an intermediate position between the L(alpha) and H(II) phases. According to their ability to lower surface tension and disintegrate at the air/water interface, the three phases thus order as L(alpha)>Pn3m>H(II). Clearly expressed threshold (minimum) bulk lipid concentrations, C(t), required for formation of stable foam bilayers from these phases, were determined and their values were found to correlate well with the bulk lipid phase behaviour. The C(t) values for L(alpha) and H(II) substantially increase with the temperature. Their Arrhenius plots, ln C(t) versus 1/ T, are linear and intersect at approximately 36-37 degrees C, coinciding with the onset of the bulk L(alpha)-->H(II) phase transition, as determined by differential scanning calorimetry. However, the C(t) value for the Pn3m phase, equal to 30 micro g/mL, was found to be constant over the whole range investigated between 20 degrees C and 50 degrees C. The horizontal C(t) versus T plot for the Pn3m phase crosses the respective plot for the L(alpha) phase at the temperature bounding from below the hysteretic loop of the L(alpha)<-->H(II) transition (approximately 26 degrees C), thus providing a certain insight about the thermodynamic stability of the Pn3m phase relative to the L(alpha) phase. The established strong effect of the particular lipid phase on the formation of monolayers and stable black foam films should be of importance in various in vitro and in vivo systems, where lipid structures are in contact with interfaces and disintegrate there to different extents.     

X-ray studies on the interaction of the antimicrobial peptide gramicidin S with microbial lipid extracts: evidence for cubic phase formation

We have investigated the effect of the interaction of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of model lipid bilayer membranes generated from the total membrane lipids of Acholeplasma laidlawii B and Escherichia coli. The A. laidlawii B membrane lipids consist primarily of neutral glycolipids and anionic phospholipids, while the E. coli inner membrane lipids consist exclusively of zwitterionic and anionic phospholipids. We show that the addition of GS at a lipid-to-peptide molar ratio of 25 strongly promotes the formation of bicontinuous inverted cubic phases in both of these lipid model membranes, predominantly of space group Pn3m. In addition, the presence of GS causes a thinning of the liquid-crystalline bilayer and a reduction in the lattice spacing of the inverted cubic phase which can form in the GS-free membrane lipid extracts at sufficiently high temperatures. This latter finding implies that GS potentiates the formation of an inverted cubic phase by increasing the negative curvature stress in the host lipid bilayer. This effect may be an important aspect of the permeabilization and eventual disruption of the lipid bilayer phase of biological membranes, which appears to be the mechanism by which GS kills bacterial cells and lysis erythrocytes.     

A comparative study of the effects of dimethylsulfoxide and glycerol on the bicontinuous cubic structure of hydrated monoolein and its phase behavior

Both dimethylsulfoxide (DMSO) and glycerol act cryoprotectants for biological systems and materials. Knowledge of molecular interactions of DMSO and glycerol with biological lipids is important for understanding of their cryoprotecitive mechanisms. In this study, the phase behavior and structures of hydrated monoolein were investigated in the presence of DMSO or glycerol, using differential scanning calorimetry (DSC) and simultaneous X-ray diffraction/DSC measurements. Based on the results obtained by this study, partial phase diagrams were constructed as a function of DMSO or glycerol concentrations and temperature. DMSO and glycerol hardly affect the enthalpy value for melting temperature of lamellar crystal phase of monoolein and the structure. On the other hand, DMSO and glycerol greatly affect the phase transformations associated with bicontinuous cubic phases of monoolein and the cubic phase structures. DMSO expands Im3m/Pn3m cubic phase co-existence region in the phase diagram and increases the lattice constant of the Pn3m monoolein cubic phase. Glycerol shows opposite effects. The present study suggests that different mechanisms act in the cryopreservation by DMSO and glycerol.     

Fusion Peptides Promote Formation of Bilayer Cubic Phases in Lipid Dispersions. An X-Ray Diffraction Study

Small angle x-ray diffraction revealed a strong influence of the N-terminal influenza hemagglutinin fusion peptide on the formation of nonlamellar lipid phases. Comparative measurements were made on a series of three peptides, a 20-residue wild-type X-31 influenza virus fusion peptide, GLFGAIAGFIENGWEGMIDG, and its two point-mutant, fusion-incompetent peptides G1E and G13L, in mixtures with hydrated phospholipids, either dipalmitoleoylphosphatidylethanolamine (DPoPE), or monomethylated dioleoyl phosphatidylethanolamine (DOPE-Me), at lipid/peptide molar ratios of 200:1 and 50:1. All three peptides suppressed the H_II phase and shifted the L_α–H_II transition to higher temperatures, simultaneously promoting formation of inverted bicontinuous cubic phases, Q_II, which becomes inserted between the L_α and H_II phases on the temperature scale. Peptide-induced Q_II had strongly reduced lattice constants in comparison to the Q_II phases that form in pure lipids. Q_II formation was favored at the expense of both L_α and H_II phases. The wild-type fusion peptide, WT-20, was distinguished from G1E and G13L by the markedly greater magnitude of its effect. WT-20 disordered the L_α phase and completely abolished the H_II phase in DOPE-Me/WT-20 50:1 dispersions, converted the Q_II phase type from Im3m to Pn3m and reduced the unit cell size from ∼38 nm for the Im3m phase of DOPE-Me dispersions to ∼15 nm for the Pn3m phase in DOPE-Me/WT-20 peptide mixtures. The strong reduction of the cubic phase lattice parameter suggests that the fusion-promoting WT-20 peptide may function by favoring bilayer states of more negative Gaussian curvature and promoting fusion along pathways involving Pn3m phase-like fusion pore intermediates rather than pathways involving H_II phase-like intermediates.     

Cubic phases in phosphatidylcholine-cholesterol mixtures: cholesterol as membrane "fusogen"

X-ray diffraction reveals that mixtures of some unsaturated phosphatidylcholines (PCs) with cholesterol (Chol) readily form inverted bicontinuous cubic phases that are stable under physiological conditions. This effect was studied in most detail for dioleoyl PC/Chol mixtures with molar ratios of 1:1 and 3:7. Facile formation of Im3m and Pn3m phases with lattice constants of 30-50 nm and 25-30 nm, respectively, took place in phosphate-buffered saline, in sucrose solution, and in water near the temperature of the Lalpha-HII transition of the mixtures, as well as during cooling of the HII phase. Once formed, the cubic phases displayed an ability to supercool and replace the initial Lalpha phase over a broad range of physiological temperatures. Conversion into stable cubic phases was also observed for mixtures of Chol with dilinoleoyl PC but not for mixtures with palmitoyl-linoleoyl PC or palmitoyl-oleoyl PC, for which only transient cubic traces were recorded at elevated temperatures. A saturated, branched-chain PC, diphytanoyl PC, also displayed a cubic phase in mixture with Chol. Unlike the PEs, the membrane PCs are intrinsically nonfusogenic lipids: in excess water they only form lamellar phases and not any of the inverted phases on their own. Thus, the finding that Chol induces cubic phases in mixtures with unsaturated PCs may have important implications for its role in fusion. In ternary mixtures, saturated PCs and sphingomyelin are known to separate into liquid-ordered domains along with Chol. Our results thus suggest that unsaturated PCs, which are excluded from these domains, could form fusogenic domains with Chol. Such a dual role of Chol may explain the seemingly paradoxical ability of cell membranes to simultaneously form rigid, low-curvature raft-like patches while still being able to undergo facile membrane fusion.     

Dynamic chain conformations in dimyristoyl glycerol-dimyristoyl phosphatidylcholine mixtures. 2H-NMR studies.

The dynamic molecular lipid chain conformations in fully hydrated dimyristoyl phosphatidylcholine (DMPC)-dimyristoyl glycerol (DMG) mixtures have been investigated by 2H-NMR spectroscopy of the individual lipid components, the sn-2 chains of which were perdeuterated or, in the case of DMG, specifically deuterated at the C-2 position. Mixtures of compositions corresponding to the three different regions of the binary phase diagram in which the fluid phase is lamellar (DMPC:DMG 70:30 mol/mol), inverted hexagonal (DMPC:DMG 45:55 and 40:60 mol/mol), or isotropic (DMPC:DMG 20:80 mol/mol) were investigated. The gel phase in all three regions of the phase diagram has a lamellar structure, with the lipid chains rotating about the molecular long axis but executing only limited angular excursions. In the fluid lamellar phase of the 70:30 mol/mol DMPC-DMG mixture the profile of segmental chain flexibility is similar to that in single-component phospholipid bilayers and is characterized by an order parameter plateau for both lipid components. The chain order of the DMPC component is greater than in bilayers of DMPC alone and is also greater than that of the DMG component. In the inverted hexagonal phase of the 45:55 mol/mol DMPC-DMG mixture the chain flexibility profile is characterized by more widely spaced segmental order parameters off the plateau region. The intrinsic degree of chain order in the inverted hexagonal phase is less than in the lamellar phase of the 70:30 mol/mol mixture, and the difference in chain order between the DMPC and DMG components is reduced relative to that in the lamellar phase. The unique conformational features at the C-2 position of the sn-2 chain that characterize bilayers of diacyl phospholipids are found also for the diacylglycerol molecules in the fluid lamellar phase and most probably also in the inverted hexagonal phase. The DMG molecules are therefore integrated in the membrane (or nonlamellar lipid phase) in a configuration that is similar to that of the phospholipids and different from the crystal structure of diacylglycerols.     

Membrane curvature stress and antibacterial activity of lactoferricin derivatives

We have studied correlation of non-lamellar phase formation and antimicrobial activity of two cationic amphipathic peptides, termed VS1-13 and VS1-24 derived from a fragment (LF11) of human lactoferricin on Escherichia coli total lipid extracts. Compared to LF11, VS1-13 exhibits minor, but VS1-24 significantly higher antimicrobial activity. X-ray experiments demonstrated that only VS1-24 decreased the onset of cubic phase formation of dispersions of E. coli lipid extracts, significantly, down to physiological relevant temperatures. Cubic structures were identified to belong to the space groups Pn3m and Im3m. Formation of latter is enhanced in the presence of VS1-24. Additionally, the presence of this peptide caused membrane thinning in the fluid phase, which may promote cubic phase formation. VS1-24 containing a larger hydrophobic volume at the N-terminus than its less active counterpart VS1-13 seems to increase curvature stress in the bilayer and alter the behaviour of the membrane significantly enhancing disruption.     

The thermotropic phase behaviour and phase structure of a homologous series of racemic beta-D-galactosyl dialkylglycerols studied by differential scanning calorimetry and X-ray diffraction

The thermotropic phase behaviour of aqueous dispersions of some synthetic 1,2-di-O-alkyl-3-O-(beta-D-galactosyl)-rac-glycerols (rac-beta-D-GalDAGs) with both odd and even hydrocarbon chain lengths was studied by differential scanning calorimetry (DSC), small-angle (SAXS) and wide-angle (WAXS) X-ray diffraction. DSC heating curves show a complex pattern of lamellar (L) and nonlamellar (NL) phase polymorphism dependent on the sample's thermal history. On cooling from 95 degrees C and immediate reheating, rac-beta-D-GalDAGs typically show a single, strongly energetic phase transition, corresponding to either a lamellar gel/liquid-crystalline (L(beta)/L(alpha)) phase transition (N< or =15 carbon atoms) or a lamellar gel/inverted hexagonal (L(beta)/H(II)) phase transition (N> or =16). At higher temperatures, some shorter chain compounds (N=10-13) exhibit additional endothermic phase transitions, identified as L/NL phase transitions using SAXS/WAXS. The NL morphology and the number of associated intermediate transitions vary with hydrocarbon chain length. Typically, at temperatures just above the L(alpha) phase boundary, a region of phase coexistence consisting of two inverted cubic (Q(II)) phases are observed. The space group of the cubic phase seen on initial heating has not been determined; however, on further heating, this Q(II) phase disappears, enabling the identification of the second Q(II) phase as Pn3 m (space group Q(224)). Only the Pn3 m phase is seen on cooling. Under suitable annealing conditions, rac-beta-D-GalDAGs rapidly form highly ordered lamellar-crystalline (L(c)) phases at temperatures above (N< or =15) or below (N=16-18) the L(beta)/L(alpha) phase transition temperature (T(m)). In the N< or =15 chain length lipids, DSC heating curves show two overlapping, highly energetic, endothermic peaks on heating above T(m); corresponding changes in the first-order spacings are observed by SAXS, accompanied by two different, complex patterns of reflections in the WAXS region. The WAXS data show that there is a difference in hydrocarbon chain packing, but no difference in bilayer dimensions or hydrocarbon chain tilt for these two L(c) phases (termed L(c1) and L(c2), respectively). Continued heating of suitably annealed, shorter chain rac-beta-D-GalDAGs from the L(c2) phase results in a phase transition to an L(alpha) phase and, on further heating, to the same Q(II) or H(II) phases observed on first heating. On reheating annealed samples with longer chain lengths, a subgel phase is formed. This is characterized by a single, poorly energetic endotherm visible below the T(m). SAXS/WAXS identifies this event as an L(c)/L(beta) phase transition. However, the WAXS reflections in the di-16:0 lipid do not entirely correspond to the reflections seen for either the L(c1) or L(c2) phases present in the shorter chain rac-beta-D-GalDAGs; rather these consist of a combination of L(c1), L(c2) and L(beta) reflections, consistent with DSC data where all three phase transitions occur within a span of 5 degrees C. At very long chain lengths (N> or =19), the L(beta)/L(c) conversion process is so slow that no L(c) phases are formed over the time scale of our experiments. The L(beta)/L(c) phase conversion process is significantly faster than that seen in the corresponding rac-beta-D-GlcDAGs, but is slower than in the 1,2-sn-beta-D-GalDAGs already studied. The L(alpha)/NL phase transition temperatures are also higher in the rac-beta-D-GalDAGs than in the corresponding rac-beta-D-GlcDAGs, suggesting that the orientation of the hydroxyl at position 4 and the chirality of the glycerol molecule in the lipid/water interface influence both the L(c) and NL phase properties of these lipids, probably by controlling the relative positions of hydrogen bond donors and acceptors in the polar region of the membrane.     

An inverse face-centered cubic phase formed by diacylglycerol-phosphatidylcholine mixtures

Fully hydrated unsaturated diacylglycerol-phosphatidylcholine mixtures are found to adopt an inverse face-centered cubic phase, of crystallographic cubic aspect 15. The same behavior is observed for either the 1,2- or 1,3-isomer of the diacylglycerol. This Q15 cubic phase, of probable space group Fd3m (Q227), occurs between an inverse hexagonal (HII) phase and an inverse micellar (L2) solution, with increasing diacylglycerol concentration, which implies that the mean curvature of the interface is more negative than that of the HII phase. This behavior is quite different from that of the more usual bicontinuous inverse cubic phases Pn3m (Q224), Im3m (Q229), and Ia3d (Q230), which normally occur between the lamellar L alpha and the HII phases. One possible structure for the Fd3m cubic phase has recently been proposed (Mariani, P., Luzzati, V., & Delacroix, H. (1988) J. Mol. Biol. 204, 165-189), consisting of tetrahedrally arranged clusters of inverse micelles surrounded by a continuous cage of tetrahedrally connected water/lipid (inverse) channels.