The effect of vitamin E on the structure of membrane lipid assemblies

The effects of vitamin E on the activity of membrane-dependent enzymes suggest that it acts indirectly by modifying some properties of the lipid host. The effects of alpha-tocopherol (alpha-T) and alpha-tocopherol hemisuccinate (alpha-THS) on phospholipid monolayer structure, curvature, and bending elasticity were examined using X-ray diffraction and the osmotic stress method. These ligands were mixed with the hexagonal phase-forming lipid, dioleoylphosphatidylethanolamine (DOPE). Increasing levels up to 50 mol% alpha-T in DOPE in excess water result in a systematic decrease in the lattice dimension. Analysis of the structural changes imposed by alpha-T shows that it contributes a spontaneous radius of curvature of -13.7 A. This unusually negative value is comparable to diacylglycerols. alpha-T does not affect the bending elasticity of these monolayers. alpha-THS in its charged form decreases membrane curvature, but in its undissociated neutral form has a qualitatively similar but reduced effect on monolayer curvature, as does alpha-T. We discuss these results in terms of the local stresses such ligands would produce in the vicinity of a membrane protein, and how one might expect proteins to respond to such stress.     

Curvature and bending constants for phosphatidylserine-containing membranes

Phosphatidylserine (PS), an anionic phospholipid of significant biological relevance, forms a multilamellar phase in water with net negative surface charge at pH 7.0. In this study we mixed dioleoylPS (DOPS) with reverse hexagonal (H(II))-forming phosphatidylethanolamine (DOPE), and used x-ray diffraction and osmotic stress to quantify its spontaneous curvature (1/R(0p)) and bending modulus (K(cp)). The mixtures were stable H(II) phases from 5 to 30 mol% PS, providing 16 wt% tetradecane (td) was also added to relieve chain-packing stress. The fully hydrated lattice dimension increased with DOPS concentration. Analysis of structural changes gave an apparent R(0p) for DOPS of +144 A; opposite in sign and relatively flat compared to DOPE (-30 A). Osmotic stress of the H(II) phases did not detect a significantly different bending modulus (K(cp)) for DOPS as compared to DOPE. At pH < or = 4.0, DOPS (with no td) adopted the H(II) phase on its own, in agreement with previous results, suggesting a reversal in curvature upon protonation of the serine headgroup. In contrast, when td was present, DOPS/td formed a lamellar phase of limited swelling whose dimension increased with pH. DOPS/DOPE/td mixtures formed H(II) phases whose dimension increased both with pH and with DOPS content. With tetradecane, estimates put 1/R(0p) for DOPS at pH 2.1 at zero. Tetradecane apparently affects the degree of dissociation of DOPS at low pH.     

The influence of lysolipids on the spontaneous curvature and bending elasticity of phospholipid membranes.

The effects of lysolipids on phospholipid layer curvature and bending elasticity were examined using x-ray diffraction and the osmotic stress method. Lysolipids with two different head groups, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and differing hydrocarbon chains were mixed with the hexagonal-forming lipid, dioleoylphosphatidylethanolamine (DOPE). With up to 30 mole% lysolipid in DOPE, the mixture maintains the inverted hexagonal (H(II)) phase in excess water, where increasing levels of lysolipid result in a systematic increase in the H(II) lattice dimension. Analysis of the structural changes imposed by lysolipids show that, opposite to DOPE itself, which has an spontaneous radius of curvature (R(0)) of -30 A, PC lysolipids add high positive curvature, with R(0) = +38 to +60 A, depending on chain length. LysoPEs, in contrast, add very small curvatures. When both polar group and hydrocarbon chains of the added lysolipid mismatch those of DOPE, the structural effects are qualitatively different from otherwise. Such mismatched lysolipids "reshape" the effective combination molecule into a longer and more cylindrical configuration compared to those lysolipids with either matching polar group or hydrocarbon chain.     

Variation of lipid membrane composition caused by strong bending

Dynamic coupling between the morphology and molecular composition of cellular membranes is crucial for formation of the intracellular organelles and transport vesicles. Most of the membrane proteins and lipids discriminate membrane curvatures. However, it remains unclear whether the curvature alone is sufficient to support heterogeneous distribution of lipids. Here we demonstrate that the curvature-driven redistribution of phospholipids, such as dioleoylphosphatidylethanolamine (DOPE), requires strong membrane bending. We used cylindrical lipid nanotubes (NTs) pulled from planar lipid membranes with lateral tension of ∼1 dyn/cm. Such high tensions forced extreme curvatures of the NT membrane, with luminal radius approaching the thickness of the lipid bilayer, 5nm. When the NT contained lipid species with high spontaneous curvature (SC), such as DOPE, we observed slow reduction of its radius. This reduction indicated the redistribution of DOPE between the inner and outer monolayers of the NT. Accordingly, the SC of DOPE was recovered from the measured changes in the radii: the SC value, calculated under the assumption that the DOPE content is coupled to the monolayer curvature, was ∼0.4 nm⁻¹, consistent with the published data. Thus, redistribution of lipids should be taken into account in calculations of composition and material properties of strongly deformed membrane structures, such as intermediate structures arising in the processes of membrane fusion and fission.     

Measured effects of diacylglycerol on structural and elastic properties of phospholipid membranes.

Diacylglycerol, a biological membrane second messenger, is a strong perturber of phospholipid planar bilayers. It converts multibilayers to the reverse hexagonal phase (HII), composed of highly curved monolayers. We have used x-ray diffraction and osmotic stress of the HII phase to measure structural dimensions, spontaneous curvature, and bending moduli of dioleoylphosphatidylethanolamine (DOPE) monolayers doped with increasing amounts of dioleoylglycerol (DOG). The diameter of the HII phase cylinders equilibrated in excess water decreases significantly with increasing DOG content. Remarkably, however, all structural dimensions at any specific water/lipid ratio that is less than full hydration are insensitive to DOG. By plotting structural parameters of the HII phase with changing water content in a newly defined coordinate system, we show that the elastic deformation of the lipid monolayers can be described as bending around a pivotal plane of constant area. This dividing surface includes 30% of the lipid volume independent of the DOG content (polar heads and a small fraction of hydrocarbon chains). As the mole fraction of DOG increases to 0.3, the radius of spontaneous curvature defined for the pivotal surface decreases from 29 A to 19 A, and the bending modulus increases from approximately 11 to 14 (+/-0.5) kT. We derive the conversion factors and estimate the spontaneous curvatures and bending moduli for the neutral surface which, unlike the pivotal plane parameters, are intrinsic properties that apply to other deformations and geometries. The spontaneous curvature of the neutral surface differs from that of the pivotal plane by less than 10%, but the difference in the bending moduli is up to 40%. Our estimate shows that the neutral surface bending modulus is approximately 9kT and practically does not depend on the DOG content.     

Curvature properties of novel forms of phosphatidylcholine with branched acyl chains.

We studied the properties of a series of phosphatidylcholine molecules with branched acyl chains. These lipids have previously been shown to have marked stimulatory effects on the side-chain cleavage activity of cytochrome P450SCC (CYP11A1), an enzyme of the inner mitochondrial membrane. The synthetic lipids used were diacyl phosphatidylcholines with the decanoyl, dodecanoyl or tetradecanoyl chain having a hexyl, octyl or decyl straight chain aliphatic branch at the 2-position. All three lipids lowered the bilayer to hexagonal phase transition temperature of dielaidoyl phosphatidylethanolamine, the lipids with longer acyl chains being more effective in this regard. As pure lipids all of the forms were found by X-ray diffraction to be predominantly in the hexagonal phase (HII) over the entire temperature range of 7-75 degrees C. The properties of the HII phase were unusual with regard to the small size of the lattice spacings and the small temperature dependence of the spacings. We used tetradecane to relieve hydrocarbon packing constraints to determine the intrinsic radius of curvature of the lipid monolayer. The elastic bending modulus was measured in the presence of tetradecane by introducing an osmotic gradient across the hexagonal phase cylinders with aqueous solutions of poly(ethylene glycol). The elastic bending modulus was found to be higher than that observed with other lipids and to increase with temperature. Both the small intrinsic radius of curvature and the high elastic bending modulus indicate that the presence of these lipids in bilayer membranes will impose a high degree of negative curvature strain.     

An Anionic Phospholipid Enables the Hydrophobic Surfactant Proteins to Alter Spontaneous Curvature

The hydrophobic surfactant proteins, SP-B and SP-C, greatly accelerate the adsorption of the surfactant lipids to an air/water interface. Previous studies of factors that affect curvature suggest that vesicles may adsorb via a rate-limiting structure with prominent negative curvature, in which the hydrophilic face of the lipid leaflets is concave. To determine if SP-B and SP-C might promote adsorption by inducing negative curvature, we used small-angle x-ray scattering to test whether the physiological mixture of the two proteins affects the radius of cylindrical monolayers in the inverse hexagonal phase. With dioleoyl phosphatidylethanolamine alone, the proteins had no effect on the hexagonal lattice constant, suggesting that the proteins fail to insert into the cylindrical monolayers. The surfactant lipids also contain ∼10% anionic phospholipids, which might allow incorporation of the cationic proteins. With 10% of the anionic dioleoyl phosphatidylglycerol added to dioleoyl phosphatidylethanolamine, the proteins induced a dose-related decrease in the hexagonal lattice constant. At 30°C, the reduction reached a maximum of 8% relative to the lipids alone at ∼1% (w/w) protein. Variation of NaCl concentration tested whether the effect of the protein represented a strictly electrostatic effect that screening by electrolyte would eliminate. With concentrations up to 3 M NaCl, the dose-related change in the hexagonal lattice constant decreased but persisted. Measurements at different hydrations determined the location of the pivotal plane and proved that the change in the lattice constant produced by the proteins resulted from a shift in spontaneous curvature. These results provide the most direct evidence yet that the surfactant proteins can induce negative curvature in lipid leaflets. This finding supports the model in which the proteins promote adsorption by facilitating the formation of a negatively curved, rate-limiting structure.     

Role of the position of unsaturation on the phase behavior and intrinsic curvature of phosphatidylethanolamines.

The bilayer-to-hexagonal phase transition temperatures (T(H)) of di-18:1(C) phosphatidylethanolamine with double bonds at positions 6, 9, and 11 are 37 degrees C, 8 degrees C, and 28 degrees C, respectively, as measured by differential scanning calorimetry and x-ray diffraction. Thus T(H) exhibits a minimum when the C=C is around position 9, similar to what has been found for the gel-to-liquid crystalline phase transition temperature in other lipids. Factors that may contribute to the dependence of T(H) on double bond position were studied by x-ray diffraction of the hexagonal phases in the presence and absence of added alkane, with or without the osmotic stress of polyethylene glycol, and over a wide temperature range. The lattice dimensions show that the intrinsic radius of lipid monolayer curvature increases as the double bond is moved toward the tail ends. A measure of the bending moduli of these lipid monolayers shows a higher value for the 9 position, and lower values for the other two. Consideration of the bilayer-to-hexagonal transition in terms of bending and interstitial energies provides a rationale for the relative values of T(H).     

Lipid polymorphism of mixtures of dioleoylphosphatidylethanolamine and saturated and monounsaturated phosphatidylcholines of various chain lengths

The L alpha-HII phase transition behavior of many lipid-water liquid crystals is dominated by the competition between the tendency to curl the lipid layers to an intrinsic radius of curvature and opposing hydrocarbon packing constraints. In particular, packing constraints can increase the free energy of the inverted hexagonal (HII) phase as compared to that of the lamellar (L alpha) phase. This is especially true where the lipid molecule is not long enough to reach into the corners of the lattice in large hexagonal structures necessitated by a large intrinsic radius of curvature. In this paper it is shown that the addition of a minor fraction long-chain lipid to a system of otherwise uniform chain composition can also relax packing constraints, thereby lowering the lamellar to hexagonal transition temperature. For the specific systems used, dioleoylphosphatidylethanolamine (di-18:1c-PE) with minor fractions of 1,2-diacyl-sn-glycero-3-phosphocholines [di-n:1c-PC (n = 14, 18, 22, and 24)], the observed HII lattices systematically increased in size with increasing chain length, suggesting that the chain length also may affect the intrinsic curvature of the mixture. These experiments demonstrate that the lipid "shape concept", which is a qualitative expression of the concept quantitatively described by the intrinsic radius of curvature, is insufficient to understand the L alpha-HII transition. It is necessary to, at least, consider the competition between curvature and packing.     

Effects of unsaturation and curvature on the transverse distribution of intramolecular dynamics of dipyrenyl lipids.

The roles of acyl chain unsaturation and curvature in the excimer formation efficiency (EFE) of site-specific conjugated pyrene molecules in lipid membranes have been investigated by steady-state and time-resolved fluorescence spectroscopy. Six 1-2-(pyrenyl-n-acyl)-phosphatidylcholine (dipy(n)PC) probes, with pyrenyl chains of varying methylene units n from 4 to 14 carbons, were incorporated separately into dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylethanolamine (DOPE) lipid membranes at 0.1 mol%. Both the excimer-to-monomer fluorescence intensity ratio and association-to-dissociation rate constant ratio of conjugated pyrenes were used to quantify EFE. At all temperatures (T = 0-30 degrees C) and for n = 4 and 6, the EFE for DOPE was always smaller than EFE for DOPC. At T < 10 degrees C (where DOPE and DOPC are in the liquid crystalline L alpha phase) and for n > 8, the EFE for curvature frustrated DOPE was significantly greater than EFE for nonfrustrated DOPC (control), and the difference increased gradually with n. At T> 18 degrees C (where DOPE is in the inverted hexagonal H(II) phase and DOPC is in the L alpha phase) and for n > 8, EFE for the curvature-relaxed DOPE was again smaller than the EFE for DOPC control. The contributions of splay conformation and internal dynamics of pyrenyl chains to EFE were examined separately using a lattice model. Our results suggest that i) the cis double bonds of the host lipid matrix strongly perturb both the conformation and dynamics of conjugated pyrenes at the specific location around n = 8, and ii) the lateral stress at the upper part (n < 8) of the curvature frustrated bilayer membranes (DOPE) may be significantly relaxed once the membrane surface adopts a favorable negative interfacial curvature.     

Rigidity and spontaneous curvature of lipidic monolayers in the presence of trehalose: a measurement in the DOPE inverted hexagonal phase

Trehalose is a sugar which plays an important protectant role in organisms against damage due to dehydration. To explore the basic molecular mechanism which governs the protective function exerted on lipid membranes, X-ray diffraction and osmotic stress experiments have been performed on l--dioleoyl-phosphatidyl-ethanolamine (DOPE) in trehalose solutions of different concentrations. In pure water, DOPE forms an inverted hexagonal (H_II) phase; in sugar solutions, a strong dehydration, which induces a large reduction of the H_II lattice parameter, has been detected, but nevertheless no phase transitions occur. Structural data, directly obtained from reconstructed electron density maps, show that the bending of the lipid monolayer induced by the sugar is coupled to changes in the DOPE molecular shape. By osmotic stress, the work required to dehydrate the monolayer has been obtained and the overall free energy described as a function of trehalose concentration. Three results should be stressed: (1) dehydration experiments performed in the presence of sugar demonstrate that the protective effect cannot be purely osmotic; (2) the pivotal surface, that location on the molecule whose area is invariant upon isothermal bending, has been analyzed by two different methods: the approach by Rand and co-workers and the approach by Templer and co-workers; in both cases its presence along the DOPE molecule has been revealed and its position estimated; (3) the spontaneous radius of curvature and the rigidity constant of the lipid monolayer, measured at the pivotal plane, changes from 3.06 nm (in pure water) to 2.82 nm (in 1.4 M trehalose), and from 0.55×10^–19 to 0.74×10^–19 J, respectively. We assume that these modifications are related to direct interactions between trehalose and DOPE that alter the interface geometry, reducing the repulsion between the polar heads. However, the increased bending rigidity also accounts for the changes of the property of the aqueous compartment, reflecting the rigidity and stiffness of the sugar matrix around and inside the lipid phase.     

X-ray diffraction study of the polymorphic behavior of N-methylated dioleoylphosphatidylethanolamine

The polymorphic phase behavior of aqueous dispersions of dioleoylphosphatidylethanolamine (DOPE) and its N-methylated analogues, DOPE-Me, DOPE-Me2, and DOPC, has been investigated by X-ray diffraction. In the fully hydrated lamellar (L alpha) phase at 2 degrees C, the major structural difference is a large increase in the interlamellar water width from DOPE to DOPE-Me, with minor increases with successive methylation. Consistent with earlier reports, inverted hexagonal (HII) phases are observed upon heating at 5-10 degrees C in DOPE and at 65-75 degrees C in DOPE-Me and are not observed to at least 85 degrees C in DOPE-Me2 or DOPC. In DOPE, the L alpha-HII transition is facile and is characterized by a relatively narrow temperature range of coexistence of L alpha and HII domains, each with long-range order. DOPE-Me exhibits complex nonequilibrium behavior below the occurrence of the HII phase: Upon heating, the L alpha lattice spontaneously disorders on a time scale of days; on cooling from the HII phase, the disorder rises on a time scale of minutes. It is shown that, in copious water, the disordered state transforms very slowly into phases with cubic symmetry. This process is assisted by the generation of small amounts of lipid degradation products. The relative magnitudes of the monolayer spontaneous radius of curvature, R0 [Kirk, G. L., Gruner, S. M., & Stein, D. L. (1984) Biochemistry 23, 1093; Gruner, S. M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3665], are inferred from the HII lattice spacings vs temperature and are shown to increase with increasing methylation. The relative magnitudes of R0 are categorized as small for DOPE, intermediate for DOPE-Me, and large for DOPC. It is suggested, and examples are used to illustrate, that small R0 lipid systems exhibit facile, low-temperature L alpha-HII transitions, intermediate R0 systems exhibit complex nonequilibrium transition behavior and are likely to form cubic phases, and large R0 systems are stable as L alpha phases. The relationship between the cubic phases and minimal periodic surfaces is discussed. It is suggested that minimal periodic surfaces represent geometries in which near constant, intermediate R0 values can be obtained concomitantly with monolayers of near constant thickness, thereby leading to equilibrium cubic phases. Thus, the relative magnitude of the spontaneous radius of curvature may be used to predict mesomorphic behavior.(ABSTRACT TRUNCATED AT 400 WORDS)     

N-succinyldioleoylphosphatidylethanolamine: structural preferences in pure and mixed model membranes

The structural preferences of the pH-sensitive phospholipid, N-succinyldioleoylphosphatidylethanolamine (N-succinyl-DOPE), have been examined alone and in mixtures with DOPE by 31P-NMR, fluorescence energy transfer, and freeze-fracture techniques. The basic polymorphic behavior of pure N-succinyl-DOPE and DOPE/N-succinyl-DOPE lipid systems and the influence of calcium and pH were investigated. It is shown that, similar to other negatively charged acidic phospholipids, N-succinyl-DOPE adopts the bilayer organization upon hydration. This structure is maintained at both pH 7.4 and 4.0 in the presence or absence of calcium. In the mixed lipid system, N-succinyl-DOPE can stabilize the non-bilayer lipid, DOPE, into a bilayer structure at both pH 7.4 and 4.0 at more than 10 mol% N-succinyl-DOPE, although a narrow 31P-NMR lineshape is observed at acidic pH values. This corresponds to the presence of smaller vesicles as shown by quasi-elastic light scattering measurements. Addition of equimolar calcium (with respect to N-succinyl-DOPE) to the DOPE/N-succinyl-DOPE systems induces the hexagonal HII phase at both pH values. In unilamellar systems with similar lipid composition the addition of Ca2+ results in membrane fusion as indicated by fluorescence energy-transfer experiments. These findings are discussed with regard to the molecular mechanism of the bilayer to hexagonal HII phase transition and membrane fusion and the utility of N-succinyl-DOPE containing pH-sensitive vesicles as drug-delivery vehicles.     

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.     

Gramicidin-induced hexagonal HII phase formation in negatively charged phospholipids and the effect of N- and C-terminal modification of gramicidin on its interaction with zwitterionic phospholipids

The effect of gramicidin on macroscopic structure of the negatively charged membrane phospholipids cardiolipin, dioleoylphosphatidylglycerol and dioleoylphosphatidylserine in aqueous dispersions was investigated and compared with the effect of gramicidin on dioleoylphosphatidylcholine. It was shown by small-angle X-ray diffraction, 31P nuclear magnetic resonance and freeze-fracture electron microscopy that in all these lipid systems gramicidin is able to induce the formation of a hexagonal HII phase. 31P-NMR measurements indicated that the extent of HII phase formation in the various lipids ranged from about 40% to 60% upon gramicidin incorporation in a molar ratio of peptide to lipid of 1 : 10. Next, the following charged analogues of gramicidin were prepared: desformylgramicidin, N-succinylgramicidin and O-succinylgramicidin. The synthesis was verified with 13C-NMR and the effect of these analogues on lipid structure was investigated. It was shown that, as with gramicidin itself, the analogues induce HII phase formation in dioleoylphosphatidylcholine, lower and broaden the bilayer-to-HII phase transition in dielaidoylphosphatidylethanolamine and form lamellar structures upon codispersion with palmitoyllysophosphatidylcholine. Differential scanning calorimetry measurements indicated that, again like gramicidin, in phosphatidylethanolamine the energy content of the gel-to-liquid-crystalline phase transition is not affected by incorporation of the analogues, whereas in phosphatidylcholine a reduction of the transition enthalpy is found. These observations were explained in terms of a similar tendency to self-associate for gramicidin and its charged analogues. The results are discussed in the light of the various factors which have been suggested to be of importance for the modulation of lipid structure by gramicidin.     

2H-nuclear magnetic resonance investigations on phospholipid acyl chain order and dynamics in the gramicidin-induced hexagonal HII phase.

The following results are reported in this paper: The interaction of gramicidin with [11,11-2H2]dioleoylphosphatidylcholine (DOPC) and [11,11-2H2]dioleoylphosphatidylethanolamine (DOPE) at different stages of hydration was studied by 2H- and 31P-nuclear magnetic resonance. In the L alpha phase in excess water the acyl chains of phosphatidylethanolamine (PE) are more ordered than phosphatidylcholine (PC) most likely as the result of the lower headgroup hydration of the former lipid. In excess water gramicidin incorporation above 5 mol % in DOPC causes a bilayer----hexagonal HII phase change. In the HII phase acyl chain order is virtually unaffected by gramicidin but the peptide restricts the fast chain motions. At low water content gramicidin cannot induce the HII phase but it markedly decreases chain order in the DOPC bilayer. Increasing water content results in separation between a gramicidin-poor and a gramicidin-rich L alpha phase with decreased order of the entire lipid molecule. Further increase in hydration reverts at low gramicidin contents the phase separation and at high gramicidin contents results in a direct change of the disordered lamellar to the hexagonal HII phase. Gramicidin also promotes HII phase formation in the PE system but interacts much less strongly with PE than with PC. The results support our hypothesis that gramicidin, by a combination of strong intermolecular attraction forces and its pronounced cone shape, both involving the four tryptophans at the COOH-terminus, has a strong tendency to organize, with the appropriate lipid, in intramembranous cylindrical structures such as is found in the HII phase.     

Nonlamellar-Phase-Promoting Colipids Enhance Segregation of Palmitoyl Ceramide in Fluid Bilayers

Ceramide is an important intermediate in sphingolipid homeostasis. We examined how colipids, with negative intrinsic curvature and which may induce curvature stress in the bilayers, affected the segregation of palmitoyl ceramide (PCer). Such colipids include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and tetra-linoleoyl cardiolipin (CL). In 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers, PCer formed ordered, gel-like domains at concentrations above 10 mol% at 23°C, as evidenced by the change in the average lifetime of the trans-parinaric acid emission. When POPE or DOPE were included in the DOPC bilayer (at 20:80 or 40:60 POPE or DOPE to DOPC, by mol), the lateral segregation of PCer was facilitated in a concentration-dependent manner, and less PCer was required for the formation of the ordered ceramide-rich domains. Inclusion of CL in the DOPE bilayer (at 10:90 or 20:80 CL to PC, by mol) also caused a similar facilitation of the lateral segregation of PCer. The PCer-rich domains formed in the presence of POPE, DOPE, or CL in DOPC bilayers were slightly more thermostable (by 2–10°C) when compared to PCer-rich domains in DOPC-only bilayers. Nonlamellar phases were not present in bilayers in which the effects of POPE or DOPE on PCer segregation were the largest, as verified by ³¹P NMR. When palmitoyl sphingomyelin was added to the different bilayer compositions at 5 mol%, relative to the phospholipids, PCer segregated into gel domains at lower concentrations (2–3 mol% PCer), and the effect of POPE on PCer segregation was eliminated. We suggest that the effects of POPE, DOPE, and CL on PCer segregation was in part influenced by their effects on membrane curvature stress and in part because of unfavorable interactions with PCer due to their unsaturated acyl chains. These lipids are abundant in mitochondrial membranes and are likely to affect functional properties of saturated ceramides in them.     

Multicomponent spectra from 31P-NMR studies of the phase equilibria in the system dioleogylphosphatidylcholine-dioleoylphosphatidylthanolamine-water

The phase equilibria in mixtures of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and water were studied by ³¹P-NMR and ²H-NMR. The chemical shift anisotropy is greater for DOPC than for DOPE (6–9 ppm in the lamellar phase). This difference can most probably be ascribed to different order parameters for the two lipid head groups. ³¹P-NMR spectra recorded from a lamellar phase formed by DOPC-DOPE-water below maximum hydration exhibit two resolved, superimposed powder spectra. The chemical shift anisotropy for both phospholipids has greater values at excess water contents than below maximum hydration, and the spectral resolution between DOPC and DOPE in the lamellar phase is strikingly diminished at excess water contents. From ³¹P-NMR spectra it is possible to observe relative differences in composition between different lipid phase existing in equilibrium. The proportion of DOPE is decreased in the lamellar phase, and is increased in the reversed hexagonal phase, when these phases exist in equilibrium.     

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)