Lipid polymorphism: a correction. The structure of the cubic phase of extinction symbol Fd-- consists of two types of disjointed reverse micelles embedded in a three-dimensional hydrocarbon matrix.

The X-ray scattering study of a cubic phase of extinction symbol Fd--, recently performed on a lipid extract (PFL) from Pseudomonas fluorescens [Mariani et al. (1990) Biochemistry 29, 6799-6810] has been extended to several other systems, all consisting of mixtures of water-miscible (MO, PC, PE, oleate) and of water-immiscible (FA, DG) lipids, plus water. In all of these systems the cubic phase was observed in the presence of excess water. Some inconsistencies observed between PFL and the other systems, the fact that in PFL one of the reflections of the cubic phase happened to coincide with the strongest reflection of the hexagonal phase, and the finding, in one of the original cubic samples of PFL kept in the cold for more than 3 years, that the intensity of one of the reflections had decreased dramatically all indicated that a nonnegligible amount of a hexagonal impurity was in fact present in the samples of PFL originally thought to contain a pure cubic phase. The intensities were corrected for that impurity and analyzed again using a pattern recognition approach based upon the axiom that the histogram of the electron density maps is invariant with respect to physical structure, when different phases are compared whose chemical composition is the same. The hexagonal phase provided the reference phase for the comparison. The moments mean value of (delta rho)n were used to compare the histograms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cubic phases of lipid-containing systems. The structure of phase Q223 (space group Pm3n). An X-ray scattering study.

The hexagonal (H) and the cubic (Q223) phases of the systems dodecyltrimethylammonium chloride-water and palmitoyllysophosphatidy choline-water have been studied by X-ray scattering techniques. The signs of the reflections of phase H were determined by a systematic study as a function of the water content, those of phase Q223 were assessed using a pattern recognition approach based upon the axiom that the histograms of the electron density maps of phases Q223 and H, extrapolated to the same concentration and properly normalized in scale and shape, are very similar to each other. In the case of phase Q223, all the sign combinations (the phi-sets) compatible with the observed reflections were generated, and each of the corresponding histograms was compared with the histogram of the map of phase H. One novelty of this work is the use of a highly sensitive criterion to estimate the similarity of the histograms, namely the distance in the six-dimensional space of the moments [mean value of (delta rho)n]1/n, for 3 greater than or equal to n greater than or equal to 8. In the two systems, the use of this criterion has led to the unambiguous choice of one electron density map. The maps show that the structure of phase Q223 consists of disjointed micelles (of type I), belonging to two different classes: those of one class are quasi-spherical in shape and are centered at the points a, those of the other class are disc-shaped and are centred at the points c. The results of this work rule out a structure formed by a cage-like distribution of rods enclosing a set of quasi-spherical micelles and is consistent with previous proposals. This is the second example, after that of phase Q227, of a micellar cubic phases in lipid-containing systems; all the known examples of phase Q223 are of type I, those of phase Q227 of type II.     

Cubic phases of lipid-containing systems. Structure analysis and biological implications

Of the six cubic phases identified so far in lipid-containing systems, the structures of only two have been determined unambiguously. We tackle the structure determination of the other four. We use for that purpose a novel pattern recognition approach, which consists of generating all the sets of phase angles (phi-sets) compatible with the observed reflections, and of screening them in a search for the "best" one. Two criteria are used for screening: both involve the parameter [(delta rho)4] (delta rho is a dimensionless function proportional to the Fourier transform of the set of observed structure factors). One is a test of smoothness, based upon the postulate that the "best" phi-set is that whose [(delta rho)4] is minimum; this criterion, equivalent to maximum entropy, is fulfilled when the system is devoid of heavy atoms, and when the polar and the hydrocarbon moieties occupy almost equal volumes. The other criterion is based upon the notion that [(delta rho)4] takes the same (or similar) values in thermodynamic phases with the same (or similar) chemical composition, whatever the structure of the phases. The validity of the two criteria is verified using numerous examples. The six cubic phases are analysed using this approach. The structure of three of them (Q230, Q224, Q229) can be described in terms of two three-dimensional networks of connected rods, mutually intertwined and unconnected: in Q230 the rods are coplanarly joined 3 by 3; in Q224 the rods are tetrahedrally joined 4 by 4; in Q229 the rods are cubically joined 6 by 6. The structures of Q212 and Q227 are related to those of Q230 and Q224, respectively; one of the two networks of rods is preserved, the other is replaced by a lattice of closed micelles. The structure of Q223 appears to consist of a cage-like continuous three-dimensional network of connected globules, coplanarly joined 3 by 3 at one end and 4 by 4 at the other, enclosing a three-dimensional lattice of closed micelles. The analogies of the structures of Q230, Q224, Q229 with the three fundamental cubic infinite periodic minimal surfaces are discussed. More interestingly, the structures of, on the one hand Q230, Q224, Q229 and of Q212, Q227, Q223 on the other, are shown to provide topological generalizations of the two paradigms of lipid organization; namely, the bilayer and the monolayer.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Phase transition from a gel to a fluid phase of cubic symmetry in dimyristoylphosphatidylcholine/myristic acid (1:2, mol/mol) bilayers

Aqueous dispersions (pH 4.0) of a 2:1 (mol/mol) mixture of myristic acid with dimyristoylphosphatidylcholine undergo a sharp transition at 45-47 degrees C from a lamellar gel phase to a fluid phase which is optically isotropic. This fluid phase gives rise to 31P-NMR spectra, and 2H-NMR spectra of the chain-deuterated components, which are also isotropic. X-ray diffraction studies of the fluid phase at 49 degrees C, reveal reflections with spacings in the ratio square root of 2: (square root of 3): square root of 4: square root of 6: square root of 8, accompanied by a strong diffuse scatter. These reflections index on a cubic lattice of primitive space group Pn3 or Pn3m, or possibly the body-centered group Im3m, with a lattice constant of 21.2 nm. The dimensions of the phase are consistent with a structure composed of two systems of tetrahedrally (octahedrally) oriented inverted lipid cylinders, found for other cubic lipid phases with Pn3m (Im3m) symmetry. At higher temperatures the cubic phase gradually converts, with increasing temperature, to a coexisting inverted hexagonal phase.     

Further evidence for closed, nonspherical aggregates in the cubic I1 phase of lysolecithin and water

Measurements of time-resolved fluorescence quenching have been performed in the binary lauroyllysophosphatidylcholine (LaLPC)/water system. The aggregation numbers, N, are determined for the micellar solution phase (N_micelle ≈ 80) and the cubic liquid crystalline I₁ phase (N_cub ≈ 90) at 298-303 K. When a quencher is present, the fluorescence decays for the hexagonal phase of the LaLPC/water system and for the bicontinuous cubic phase of monooleoylglycerol/water system are nonexponential, as expected for phase structures having long-range continuous apolar regions. Nuclear magnetic resonance (NMR) measurements of the lipid translational diffusion conclusively show that the cubic I₁ phase consists of closed micelles. NMR spectra of ³¹P obtained at 202.4 MHz of this cubic phase exhibit a characteristic line shape, which is compatible with a phase structure containing short nonspherical micelles. A comparison between electron spin resonance (ESR) spin-label spectra recorded for a micellar solution and the cubic phases of the LaLPC and monooleoylglycerol systems are also shown to support a structure of closed micelles in the cubic I₁ phase of the lysolecithin system.     

Phase equilibria of mixtures of plant galactolipids. The formation of a bicontinuous cubic phase

The chloroplast galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were isolated from wheat leaves. The phase equilibria of galactolipid-water systems with MGDG / DGDG molar ratios equal to 0:1, 1:2, 1.2:1, 2:1 and 1:0 were investigated, using nuclear magnetic resonance (NMR) methods. MGDG and DGDG form reversed hexagonal and lamellar phases, respectively, at temperatures between 10 and 40°C at all water contents studied (up to about 14 mol ²H₂O per mol lipid). The galactolipid mixtures show a complex phase forming reversed hexagonal, lamellar and reversed cubic phases, depending on water content and temperature. It was found that the water hydration is similar for the lamellar and hexagonal phases formed by DGDG and MGDG, respectively. The non-lamellar phase areas increase with increasing content of MGDG. Small-angle X-ray measurements show that the cubic phase belongs to the Ia3d space group. From translational diffusion studies by NMR it is concluded that the structure of this cubic phase is bicontinuous.     

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.     

Hydrophobic lipid additives affect membrane stability and phase behavior of N-monomethyldioleoylphosphatidylethanolamine.

The rate of formation of high-curvature intermediates or disordered cubic phases in N-methyldioleoylphosphatidylethanolamine (N-methyl-DOPE) dispersions with or without additives was studied by 31P NMR spectroscopy. In N-methyl-DOPE dispersions, both the L alpha liquid-crystalline phase and the hexagonal HII phase convert into phases of high curvature giving rise to isotropic 31P NMR resonances. Addition of the bilayer destabilizers 1,2-diolein, 1,3-diolein, or eicosane lowers the threshold temperature of the isotropic phase. The isotropic threshold temperature is strongly correlated with the L alpha-HII phase transition temperature (TH). The addition of hexagonal phase promoters does not change the rate of formation of the isotropic phase at a temperature shifted by a fixed amount below TH. However, the formation of "isotropic" phases from the additive-stabilized hexagonal phase is slow compared to that observed in pure N-methyl-DOPE lipid dispersions. Membrane leakage and fusion are promoted by the dioleins and well as by eicosane, but changes in the rates of these processes do not correlate well with the extent of formation of isotropic phases. All three additives have similar effects on phase behavior and on vesicle leakage and fusion. These similarities occur despite the fact that eicosane is believed to partition differently into the membrane than diolein. In addition to the general similarities in the effects of the two diolein isomers, 1,2-diolein is somewhat more potent in promoting the hexagonal phase and in increasing rates of leakage and fusion than is 1,3-diolein.     

The gaussian curvature elastic modulus of N-monomethylated dioleoylphosphatidylethanolamine: relevance to membrane fusion and lipid phase behavior

The energy of intermediates in fusion of phospholipid bilayers is sensitive to kappa(m), the saddle splay (Gaussian curvature) elastic modulus of the lipid monolayers. The value kappa(m) is also important in understanding the stability of inverted cubic (Q(II)) and rhombohedral (R) phases relative to the lamellar (L(alpha)) and inverted hexagonal (H(II)) phases in phospholipids. However, kappa(m) cannot be measured directly. It was previously measured by observing changes in Q(II) phase lattice dimensions as a function of water content. Here we use observations of the phase behavior of N-mono-methylated dioleoylphosphatidylethanolamine (DOPE-Me) to determine kappa(m). At the temperature of the L(alpha)/Q(II) phase transition, T(Q), the partial energies of the two phases are equal, and we can express kappa(m) in terms of known lipid monolayer parameters: the spontaneous curvature of DOPE-Me, the monolayer bending modulus kappa(m), and the distance of the monolayer neutral surface from the bilayer midplane, delta. The calculated ratio kappa(m)/kappa(m) is -0.83 +/- 0.08 at T(Q) approximately 55 degrees C. The uncertainty is due primarily to uncertainty in the value of delta for the L(alpha) phase. This value of kappa(m)/kappa(m) is in accord with theoretical expectations, including recent estimates of the value required to rationalize observations of rhombohedral (R) phase stability in phospholipids. The value kappa(m) substantially affects the free energy of formation of fusion intermediates: more energy (tens of k(B)T) is required to form stalks and fusion pores (ILAs) than estimated solely on the basis of the bending elastic energy. In particular, ILAs are much higher in energy than previously estimated. This rationalizes the action of fusion-catalyzing proteins in stabilizing nascent fusion pores in biomembranes; a function inferred from recent experiments in viral systems. These results change predictions of earlier work on ILA and Q(II) phase stability and L(alpha)/Q(II) phase transition mechanisms. To our knowledge, this is the first determination of the saddle splay (Gaussian) modulus in a lipid system consisting only of phospholipids.     

Induction of non-lamellar lipid phases by antimicrobial peptides: a potential link to mode of action

Antimicrobial peptides are naturally produced by numerous organisms including insects, plants and mammals. Their non-specific mode of action is thought to involve the transient perturbation of bacterial membranes but the molecular mechanism underlying the rearrangement of the lipid molecules to explain the formation of pores and micelles is still poorly understood. Biological membranes mostly adopt planar lipid bilayers; however, antimicrobial peptides have been shown to induce non-lamellar lipid phases which may be intimately linked to their proposed mechanisms of action. This paper reviews antimicrobial peptides that alter lipid phase behavior in three ways: peptides that induce positive membrane curvature, peptides that induce negative membrane curvature and peptides that induce cubic lipid phases. Such structures can coexist with the bilayer structure, thus giving rise to lipid polymorphism induced upon addition of antimicrobial peptides. The discussion addresses the implications of induced lipid phases for the mode of action of various antimicrobial peptides.     

Mechanism of the lamellar/inverse hexagonal phase transition examined by high resolution x-ray diffraction

For the first time the electron density of the lamellar liquid crystalline as well as of the inverted hexagonal phase could be retrieved at the transition temperature. A reliable decomposition of the d-spacings into hydrophobic and hydrophilic structure elements could be performed owing to the presence of a sufficient number of reflections. While the hydrocarbon chain length, d(C), in the lamellar phase with a value of 14.5 A lies within the extreme limits of the estimated chain length of the inverse hexagonal phase 10 A < d(C) < 16 A, the changes in the hydrophilic region vary strongly. During the lamellar-to-inverse hexagonal phase transition the area per lipid molecule reduces by approximately 25%, and the number of water molecules per lipid increases from 14 to 18. On the basis of the analysis of the structural components of each phase, the interface between the coexisting mesophases between 66 and 84 degrees C has been examined in detail, and a model for the formation of the first rods in the matrix of the lamellar phospholipid stack is discussed. Judging from the structural relations between the inverse hexagonal and the lamellar phase, we suggest a cooperative chain reaction of rod formation at the transition midpoint, which is mainly driven by minimizing the interstitial region.     

Hydrophobic Surfactant Proteins Induce a Phosphatidylethanolamine to Form Cubic Phases

The hydrophobic surfactant proteins SP-B and SP-C promote rapid adsorption of pulmonary surfactant to an air/water interface. Previous evidence suggests that they achieve this effect by facilitating the formation of a rate-limiting negatively curved stalk between the vesicular bilayer and the interface. To determine whether the proteins can alter the curvature of lipid leaflets, we used x-ray diffraction to investigate how the physiological mixture of these proteins affects structures formed by 1-palmitoyl-2-oleoyl phosphatidylethanolamine, which by itself undergoes the lamellar-to-inverse hexagonal phase transition at 71°C. In amounts as low as 0.03% (w:w) and at temperatures as low as 57°C, the proteins induce formation of bicontinuous inverse cubic phases. The proteins produce a dose-related shift of diffracted intensity to the cubic phases, with minimal evidence of other structures above 0.1% and 62°C, but no change in the lattice-constants of the lamellar or cubic phases. The induction of the bicontinuous cubic phases, in which the individual lipid leaflets have the same saddle-shaped curvature as the hypothetical stalk-intermediate, supports the proposed model of how the surfactant proteins promote adsorption.     

Phase diagram of soybean phosphatidylcholine-diacylglycerol-water studied by x-ray diffraction and 31P- and pulsed field gradient 1H-NMR: evidence for reversed micelles in the cubic phase.

The phase equilibria of the system soybean phosphatidylcholine, diacylglycerol, and water has been determined using a combination of classical methods together with x-ray diffraction and NMR techniques. In particular, the extent of the phase regions of the lamellar, the reversed hexagonal, and the cubic phases have been determined. By pulsed field gradient 1H-NMR, the diffusion coefficients of all three components in a cubic phase composed of soybean phosphatidylcholine, diacylglycerol, and heavy water have been determined at 25 and 59 degrees C and also for the corresponding cubic phase composed of the chemically more well defined synthetic components 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoylglycerol (DOG), and heavy water. The extension of the phase region of the cubic phase did not seem to change appreciably for the two ternary systems studied. The translational diffusion coefficient of DOPC in this cubic phase is more than an order of magnitude smaller (3 x 10(-13) m2 s-1, 59 degrees C) than the lateral diffusion coefficient of DOPC in an oriented lipid bilayer (5 x 10(-12) m2 s-1, 35 degrees C), whereas the diffusion coefficients of water and DOG were found to be about two orders of magnitude larger than DOPC at 59 degrees C. It is concluded that the cubic phase is built built up of closed reversed micelles in accordance with the suggestion from previous x-ray diffraction studies.     

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.     

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)     

Effect of bile salts on monolayer curvature of a phosphatidylethanolamine/water model membrane system.

A partial phase diagram of the ternary system dioleoylphosphatidylethanolamine (DOPE)/sodium cholate/water has been determined using 31P Nuclear Magnetic Resonance (NMR) spectroscopy. In the absence of cholate, it is well known that the DOPE/water system forms a reversed hexagonal (HII) phase. We have found that addition of even small amounts of cholate to the DOPE/water system leads to a transition to a lamellar (L alpha) phase. At higher cholate concentrations, a cubic (I) phase (low water content) or a micellar solution (L1) phase (high water content) is present. Thus, cholate molecules have a strong tendency to alter the lipid monolayer curvature. Increasing the concentration of cholate changes the curvature of DOPE from negative (HII phase), through zero (L alpha phase), and finally to a phase of positive curvature (micellar solution). This observation can be rationalized in terms of the molecular structure of cholate, which is amphipathic and has one hydrophobic and one hydrophilic side of the steroid ring system. The cholate molecules have a tendency to lie flat on the lipid aggregate surface, thereby increasing the effective interfacial area of the polar head groups, and altering the curvature free energy of the system.     

Evidence of cholesterol accumulated in high curvature regions: implication to the curvature elastic energy for lipid mixtures

Recent experiments suggested that cholesterol and other lipid components of high negative spontaneous curvature facilitate membrane fusion. This is taken as evidence supporting the stalk-pore model of membrane fusion in which the lipid bilayers go through intermediate structures of high curvature. How do the high-curvature lipid components lower the free energy of the curved structure? Do the high-curvature lipid components modify the average spontaneous curvature of the relevant monolayer, thereby facilitate its bending, or do the lipid components redistribute in the curved structure so as to lower the free energy? This question is fundamental to the curvature elastic energy for lipid mixtures. Here we investigate the lipid distribution in a monolayer of a binary lipid mixture before and after bending, or more precisely in the lamellar, hexagonal, and distorted hexagonal phases. The lipid mixture is composed of 2:1 ratio of brominated di18:0PC and cholesterol. Using a newly developed procedure for the multiwavelength anomalous diffraction method, we are able to isolate the bromine distribution and reconstruct the electron density distribution of the lipid mixture in the three phases. We found that the lipid distribution is homogenous and uniform in the lamellar and hexagonal phases. But in the distorted hexagonal phase, the lipid monolayer has nonuniform curvature, and cholesterol almost entirely concentrates in the high curvature region. This finding demonstrates that the association energies between lipid molecules vary with the curvature of membrane. Thus, lipid components in a mixture may redistribute under conditions of nonuniform curvature, such as in the stalk structure. In such cases, the spontaneous curvature depends on the local lipid composition and the free energy minimum is determined by lipid distribution as well as curvature.     

Induction of a hexagonal phase in phospholipid-surfactant bilayers

The possibility of modifying the curvature of lipid bilayers by mixing them with additives is demonstrated and the evolution of geometrical parameters with composition is discussed. X-ray diffraction patterns of the POPC/C₁₂EO₂/²H₂O system were observed as a function of the relative humidity. The formation of an unexpected hexagonal phase indicates peculiar behaviour in these mixtures. The cylinder radius of this phase is considerably smaller than previously observed in cubic phases. A discussion of the head group interactions is presented. We have also been able to show that the uptake of water by the L _β gel phase is higher as a single phase than in the L _β +H_II two phase region. The water content is important for the stabilization of H_II phase and determination of its characteristic dimensions. However, it is argued that the interaction between the surfactant and the lipid is the key factor for its formation and that the EO₂ head groups displace water from the inner parts of the polar region of the mesogenic units. Received: 22 September 1997 / Revised version: 31 December 1997 / Accepted: 10 February 1998