Inverted micellar intermediates and the transitions between lamellar, cubic, and inverted hexagonal amphiphile phases. III. Isotropic and inverted cubic state formation via intermediates in transitions between L alpha and HII phases

Inverted cubic and isotropic phases have been observed in phospholipid and glycolipid systems. These phases exhibit characteristic morphologies in freeze-fracture electron micrographs, isotropic 31P-NMR resonances and (in some cases) cubic X-ray diffraction patterns. It is proposed here that these phases may form from the same intermediates that are involved in lamellar/inverted hexagonal (L alpha/HII) phase transitions, and that it is possible that these cubic and isotropic phases are metastable. According to a kinetic theory of L alpha/HII phase transitions, intermediates in such transitions can form structures known as interlamellar attachments (ILAs). It is shown that ILAs should form in large numbers during L alpha/HII transitions in systems like those reported to form inverted cubic or isotropic structures. ILAs cannot readily assemble into either the HII phase or well-ordered arrays of L alpha phase bilayers, and represent a kinetic trap for intermediates in L alpha/HII transitions (although it is possible that they are marginally more stable in a thermodynamic sense than the L alpha phase in a small temperature range below TH). It is also shown that arrays of ILAs should form metastable arrays with the same morphology and isotropic 31P-NMR resonances that are observed in isotropic and inverted cubic states. In particular, under some circumstances ILAs will assemble into a structure identical to the bicontinuous inverted cubic phase previously described in monoglycerides and very similar in morphology to structures observed in phospholipid systems. Finally, since isotropic and cubic states form from ILAs, which also can mediate fusion of unilamellar vesicles, unilamellar vesicles should fuse to at least some extent under the same conditions in which multilamellar samples of the same lipid form isotropic or inverted cubic states. This correlation has been observed.     

Inverted micellar intermediates and the transitions between lamellar, cubic, and inverted hexagonal lipid phases. II. Implications for membrane-membrane interactions and membrane fusion.

Results of a kinetic model of thermotropic L alpha----HII phase transitions are used to predict the types and order-of-magnitude rates of interactions between unilamellar vesicles that can occur by intermediates in the L alpha----HII phase transition. These interactions are: outer monolayer lipid exchange between vesicles; vesicle leakage subsequent to aggregation; and (only in systems with ratios of L alpha and HII phase structural dimensions in a certain range or with unusually large bilayer lateral compressibilities) vesicle fusion with retention of contents. It was previously proposed that inverted micellar structures mediate membrane fusion. These inverted micellar structures are thought to form in all systems with such transitions. However, I show that membrane fusion probably occurs via structures that form from these inverted micellar intermediates, and that fusion should occur in only a sub-set of lipid systems that can adopt the HII phase. For single-component phosphatidylethanolamine (PE) systems with thermotropic L alpha----HII transitions, lipid exchange should be observed starting at temperatures several degrees below TH and at all higher temperatures, where TH is the L alpha----HII transition temperature. At temperatures above TH, the HII phase forms between apposed vesicles, and eventually ruptures them (leakage). In most single-component PE systems, fusion via L alpha----HII transition intermediates should not occur. This is the behavior observed by Bentz, Ellens, Lai, Szoka, et al. in PE vesicle systems. Fusion is likely to occur under circumstances in which multilamellar samples of lipid form the so-called "inverted cubic" or "isotropic" phase. This is as observed in the mono-methyl DOPE system (Ellens, H., J. Bentz, and F. C. Szoka. 1986. Fusion of phosphatidylethanolamine containing liposomes and the mechanism of the L alpha-HII phase transition. Biochemistry. In press.) In lipid systems with L alpha----HII transitions driven by cation binding (e.g., Ca2+-cardiolipin), fusion should be more frequent than in thermotropic systems.     

Inverted micellar structures in bilayer membranes. Formation rates and half-lives.

Two sorts of inverted micellar structures have previously been proposed to explain morphological and 31P-NMR observations of bilayer systems. These structures only form in systems with components that can adopt the inverse hexagonal (HII) phase. LIP (lipidic particles) are intrabilayer structures, whereas IMI (inverted micellar intermediates) are structures that form between apposed bilayers. Here, we calculate the formation rates and half-lives of these structures to determine which (or if either) of these proposed structures is a likely explanation of the data. Calculations for the egg phosphatidylethanolamine and the Ca+-cardiolipin systems show that IMI form orders of magnitude faster than LIP, which should form slowly, if at all. This result is probably true in general, and indicates that "lipidic particle" electron micrograph images probably represent interbilayer structures, as some have previously proposed. It is shown here that IMI are likely intermediates in the lamellar----HII phase transitions and in the process of membrane fusion in some systems. The calculated formation rates, half-lives, and vesicle-vesicle fusion rates are in agreement with this observation.     

Effects of cholesterol on the structural transitions induced by diacylglycerol in phosphatidylcholine and phosphatidylethanolamine bilayer systems

The transient membrane lipid diacylglycerol (DG) is known to modify and destabilize phospholipid bilayers and can lead to the formation of nonbilayer structures. Since cholesterol forms a major fraction of many plasma membranes, we have investigated how it modifies the structural effects of DG on bilayers of egg phosphatidylcholine (PC) and egg phosphatidylethanolamine (PE). We view these systems as modelling the behaviour of local, DG-containing sites in membranes. Using X-ray diffraction, we have characterized the lamellar (L alpha) and inverse hexagonal (HII) structures that these ternary lipid mixtures form in excess aqueous solution. As the DG level increases, the lipid progresses from a single L alpha structure to a mixture of L alpha and HII, and then to a pure HII structure. This allows determination of the DG levels at which the HII transition begins, which we interpret as those levels that destabilize bilayers. In both PC and PE bilayers, the presence of 30 mol% cholesterol reduces the amounts of DG required to destabilize the bilayer structure. The destabilization can be translated into the number of neighbouring lipid molecules that a DG molecule perturbs, and of bilayer areas that it affects. The data show that the presence of cholesterol greatly enhances the perturbing effects of DG. We examine the possible role of DG in enzyme activation and membrane fusion.     

Lamellar/inverted cubic (L alpha/QII) phase transition in N-methylated dioleoylphosphatidylethanolamine

Inverted cubic (QII) phases form in hydrated N-methylated dioleoylphosphatidylethanolamine (DOPE-Me). Previous work indicated that QII phases in this and other systems might be metastable structures. Whether or not QII phases are stable has important implications for models of the factors determining the relative stability of bilayer and nonbilayer phases and of the mechanisms of transitions between those phases. Here, using X-ray diffraction and very slow scan rate differential scanning calorimetry (DSC), we show that thermodynamically stable QII phases form slowly during incubation of multilamellar samples of DOPE-Me at constant temperature. The equilibrium L alpha/QII phase transition temperature is 62.2 +/- 1 degree C. The transition enthalpy is 174 +/- 34 cal/mol, about two-thirds of the L alpha/HII transition enthalpy observed at faster scan rates. This implies that the curvature free energy of lipids in QII phases is substantially lower than in L alpha phases and that this reduction is substantial compared to the reduction achieved in the HII phase. The L alpha/QII transition is slow and is not reliably detected with DSC until the temperature scan rate is reduced to ca. 1 degrees C/h. At faster scan rates, the HII phase forms at a reproducible temperature of 66 degrees C. This HII phase is metastable until ca. 72-79 degrees C, where the equilibrium QII/HII transition seems to occur. These results, as well as the induction of QII phases in similar systems by temperature cycling (observed by others), are consistent with a theory of L alpha/QII/HII transition mechanisms proposed earlier (Siegel, 1986c).     

The influence of phase transitions in phosphatidylethanolamine models on the activity of violaxanthin de-epoxidase

In the present study, the influence of the phospholipid phase state on the activity of the xanthophyll cycle enzyme violaxanthin de-epoxidase (VDE) was analyzed using different phosphatidylethanolamine species as model lipids. By using (31)P NMR spectroscopy, differential scanning calorimetry and temperature dependent enzyme assays, VDE activity could directly be related to the lipid structures the protein is associated with. Our results show that the gel (L beta) to liquid-crystalline (L alpha) phase transition in these single lipid component systems strongly enhances both the solubilization of the xanthophyll cycle pigment violaxanthin in the membrane and the activity of the VDE. This phase transition has a significantly stronger impact on VDE activity than the transition from the L alpha to the inverted hexagonal (HII) phase. Especially at higher temperatures we found increased VDE reaction rates in the presence of the L alpha phase compared to those in the presence of HII phase forming lipids. Our data furthermore imply that the HII phase is better suited to maintain high VDE activities at lower temperatures.     

Kinetics of the lamellar and hexagonal phase transitions in phosphatidylethanolamine. Time-resolved x-ray diffraction study using a microwave-induced temperature jump.

The kinetics of the thermotropic lamellar gel (L beta')/lamellar liquid crystal (L alpha) and L alpha/inverted hexagonal (HII) phase transitions in fully hydrated dihexadecylphosphatidylethanolamine (DHPE) have been studied. Measurements were made by using time-resolved x-ray diffraction (TRXRD) to monitor progress of the transitions. In these studies microwave energy at 2.5 GHz was used to increase the sample temperature rapidly and uniformly through the phase transition regions. The L beta'/L alpha and L alpha/HII transitions of DHPE were examined under active microwave heating and passive cooling. The transitions were found to be repeatable and reversible, and to have an upper bound on the time required to complete the transition of less than 3 s. Regardless of the direction of the transition, both phase transitions appeared to be two-state with no accumulation of intermediates to within the sensitivity limits of the TRXRD method. The rate and amplitude of the temperature jump can be controlled by regulating microwave radiation input power. A temperature jump rate of 29 degrees C/s was obtained at a final microwave power setting of 120 W. Comparisons between previously reported fluid flow (Caffrey, M. 1985. Biochemistry. 24:4826-4844) and microwave heating studies suggest that the determination of limiting transit times will require faster heating.     

Effect of the chirality of the glycerol backbone on the bilayer and nonbilayer phase transitions in the diastereomers of di-dodecyl-beta-D-glucopyranosyl glycerol.

We have studied the physical properties of aqueous dispersions of 1,2-sn- and 2,3-sn-didodecyl-beta-D-glucopyranosyl glycerols, as well as their diastereomeric mixture, using differential scanning calorimetry and low angle x-ray diffraction. Upon heating, both the chiral lipids and the diastereomeric mixture exhibit characteristically energetic L beta/L alpha phase transitions at 31.7-32.8 degrees C and two or three weakly energetic thermal events between 49 degrees C and 89 degrees C. In the diastereomeric mixture and the 1,2-sn glycerol derivative, these higher temperature endotherms correspond to the formation of, and interconversions between, several nonlamellar structures and have been assigned to L alpha/QIIa, QIIa/QIIb, and QIIb/HII phase transitions, respectively. The cubic phases QIIa and QIIb, whose cell lattice parameters are strongly temperature dependent, can be identified as belonging to space groups Ia3d and Pn3m/Pn3, respectively. In the equivalent 2,3-sn glucolipid, the QIIa phase is not observed and only two transitions are seen at 49 degrees C and 77 degrees C, which are identified as L alpha/QIIb and QIIb/HII phase transitions, respectively. These phase transitions temperatures are some 10 degrees C lower than those of the corresponding phase transitions observed in the diastereomeric mixture and the 1,2-sn glycerol derivative. On cooling, all three lipids exhibit a minor higher temperature exothermic event, which can be assigned to a HII/QIIb phase transition. An exothermic L alpha/L beta phase transition is observed at 30-31 degrees C. A shoulder is sometimes discernible on the high temperature side of the L alpha/L beta event, which may originate from a QIIb/L alpha phase transition prior to the freezing of the hydrocarbon chains. None of the lipids show evidence of a QIIa phase on cooling. No additional exothermic transitions are observed on further cooling to -3 degrees C. However, after nucleation at 0 degrees C followed by a short period of annealing at 22 degrees C, the 1,2-sn glucolipid forms an Lc phase that converts to an L alpha phase at 39.5 degrees C on heating. Neither the diastereomeric mixture nor the 2,3-sn glycerol derivative shows such behavior even after extended periods of annealing. Our results suggest that the differences in the phase behavior of these glycolipid isomers may not be attributable to headgroup size per se, but rather to differences in the stereochemistry of the lipid polar/apolar interfacial region, which consequently effects hydrogen-bonding, hydration, and the hydrophilic/hydrophobic balance.     

Membrane fusion and inverted phases

We have found a correlation between liposome fusion kinetics and lipid phase behavior for several inverted phase forming lipids. N-Methylated dioleoylphosphatidylethanolamine (DOPE-Me), or mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC), will form an inverted hexagonal phase (HII) at high temperatures (above TH), a lamellar phase (L alpha) at low temperatures, and an isotropic/inverted cubic phase at intermediate temperatures, which is defined by the appearance of narrow isotropic 31P NMR resonances. The phase behavior has been verified by using high-sensitivity DSC, 31P NMR, freeze-fracture electron microscopy, and X-ray diffraction. The temperature range over which the narrow isotropic resonances occur is defined as delta TI, and the range ends at TH. Extruded liposomes (approximately 0.2 microns in diameter) composed of these lipids show fusion and leakage kinetics which are strongly correlated with the temperatures of these phase transitions. At temperatures below delta TI, where the lipid phase is L alpha, there is little or no fusion, i.e., mixing of aqueous contents, or leakage. However, as the temperature reaches delta TI, there is a rapid increase in both fusion and leakage rates. At temperatures above TH, the liposomes show aggregation-dependent lysis, as the rapid formation of HII phase precursors disrupts the membranes. We show that the correspondence between the fusion and leakage kinetics and the observed phase behavior is easily rationalized in terms of a recent kinetic theory of L alpha/inverted phase transitions. In particular, it is likely that membrane fusion and the L alpha/inverted cubic phase transition proceed via a common set of intermembrane intermediates.     

Physical properties of glycosyl diacylglycerols. 1. Calorimetric studies of a homologous series of 1,2-di-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols

The polymorphic phase behavior of aqueous dispersions of a homologous series of 1,2-di-O-acyl-3-O-(alpha-D-glucopyranosyl)-sn-glycerols was studied by differential scanning calorimetry. At fast heating rates unannealed samples of these lipids exhibit a strongly energetic transition, which has been identified as a lamellar gel/liquid crystalline (L beta/L alpha) phase transition (short- and medium-chain compounds) or a lamellar gel to inverted hexagonal (L beta/HII) phase transition (long-chain compounds) by X-ray diffraction studies (Sen et al., 1990). At still higher temperatures, some of the lipids that form lamellar liquid-crystalline phases exhibit an additional transition, which has been identified as a transition to an inverted nonbilayer phase by X-ray diffraction studies. The lamellar gel phase formed on initial cooling of these lipids is a metastable structure, which, when annealed under appropriate conditions, transforms to a more stable lamellar gel phase, which has been identified as a poorly hydrated crystal-like phase with tilted acyl chains by X-ray diffraction measurements (Sen et al., 1990). With the exception of the di-19:0 homologue, the crystalline phases of these lipids are stable to temperatures higher than those at which their L beta phases melt and, as a result, they convert directly to L alpha or HII phases on heating. Our results indicate that the length of the acyl chain affects both the kinetic and thermodynamic properties of the crystalline phases of these lipids as well as the type of nonbilayer phase that they form. Moreover, when compared with the beta-anomers, these alpha-D-glucosyl diacylglycerols are more prone to form ordered crystalline gel phases at low temperatures and are somewhat less prone to form nonbilayer phases at elevated temperatures. Thus the physical properties of glucolipids (and possibly all glycolipids) are very sensitive to the nature of the anomeric linkage between the sugar headgroup and the glycerol backbone of the lipid molecule. We suggest that this is, in part, due to a change in orientation of the glucopyranosyl ring relative to the bilayer surface, which in turn affects the way(s) in which the sugar headgroups interact with each other and with water.     

Thermotropic and barotropic phase transitions of N-methylated dipalmitoylphosphatidylethanolamine bilayers

In order to understand the effect of polar head group modification on the thermotropic and barotropic phase behavior of phospholipid bilayer membranes, the phase transitions of dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidyl-N-methylethanolamine (DPMePE), dipalmitoylphosphatidyl-N,N-dimethylethanolamine (DPMe2PE) and dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were observed by differential scanning calorimetry and high-pressure optical methods. The temperatures of the so-called main transition from the gel (L(beta)) or ripple gel (P(beta)') phase to the liquid crystalline (L(alpha)) phase were almost linearly elevated by applying pressure. The slope of the temperature-pressure boundary, dT/dp, was in the range of 0.220-0.264 K MPa(-1) depending on the number of methyl groups in the head group of lipids. The main-transition temperatures of N-methylated DPPEs decreased with increasing size of head group by stepwise N-methylation. On the other hand, there was no significant difference in thermodynamic quantities of the main transition between the phospholipids. With respect to the transition from the subgel (L(c)) phase to the lamellar gel (L(beta) or L(beta)') phase, the transition temperatures were also elevated by applying pressure. In the case of DPPE bilayer the L(c)/L(beta) transition appeared at a pressure higher than 21.8 MPa. At a pressure below 21.8 MPa the L(c)/L(alpha) transition was observed at a temperature higher than the main-transition temperature. The main (L(beta)/L(alpha)) transition can be recognized as the transformation between metastable phases in the range from ambient pressure to 21.8 MPa. Polymorphism in the gel phase is characteristic of DPPC bilayer membrane unlike other lipid bilayers used in this study: the L(beta)', P(beta)' and pressure-induced interdigitated gel (L(beta)I) phases were observed only in the DPPC bilayer. Regarding the bilayers of DPPE, DPMePE and DPMe2PE, the interdigitation of acyl chain did not appear even at pressures as high as 200 MPa.     

Polymorphic phase behaviour of dilinoleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine mixtures. Structural changes between hexagonal, cubic and bilayer phases

The polymorphic phase behaviour of dilinoleoylphosphatidyethanolamine (DLPE) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) is investigated by freeze-fracture electron microscopy, X-ray diffraction and 31P-NMR. The structures at 5% or less POPC are predominantly inverted hexagonal (HII), whereas at 15% or more POPC, the structure is mostly bilayer (L), interrupted by defects (lipidic particles). A cubic phase structure is observed in the transition range between H and L phases; the cubic arrangement deteriorates at higher temperatures into an amorphous aggregate of spherical units. Both cubic and amorphous structures contribute to the isotropic 31P resonance, with no preference for PC or PE partitioning in the isotropic motion as observed by high resolution NMR. The existence of the cubic phase seems to depend cirtically on the homogeneity and the degree unsaturation of the phospholipids.     

Effect of acyl chain composition on salt-induced lamellar to inverted hexagonal phase transitions in cardiolipin

Salt-induced fluid lamellar (L alpha) to inverted hexagonal (HII) phase transitions have been studied in diphosphatidylglycerols (cardiolipins) with different acyl chain compositions, using 31P nuclear magnetic resonance (NMR) spectroscopy. Cardiolipins with four myristoyl chains, tetramyristoyl cardiolipin (TMCL), and with four oleoyl chains, tetraoleoyl cardiolipin (TOCL), were synthesized chemically. TMCL was found to undergo a thermotropic lamellar gel to lamellar liquid-crystalline phase transition at 33-35 degrees C. This lipid exhibited an axially symmetric 31P-NMR spectrum corresponding to a lamellar phase at all NaCl concentrations between 0 and 6 M. In the case of TOCL, formation of an HII phase was induced by salt concentrations of 3.5 M NaCl or greater. These observations, taken together with earlier findings that bovine heart cardiolipin aqueous dispersions adopt an HII phase at salt concentrations of 1.5 M NaCl or greater, indicate that increasing unsaturation and length of the acyl chains favour formation of the HII phase in diphosphatidylglycerols.     

Comparison of the orientational order of lipid chains in the L alpha and HII phases

The orientational order profile has been determined by using deuterium nuclear magnetic resonance (2H NMR) for POPE in the lamellar liquid-crystalline (L alpha) and the hexagonal (HII) phases and is shown to be sensitive to the symmetry of the lipid phase. In the HII phase, as compared to the L alpha phase, the acyl chains are characterized by a greater motional freedom, and the orientational order is distributed more uniformly along the lipid acyl chain. This is consistent with a change from a cylindrical to a wedge-shaped space available for the lipid chain. 2H NMR studies of POPE dispersions containing tetradecanol or decane, both of which can induce HII phase structure, show very different behavior. Tetradecanol appears to align with the phospholipid chains and experience the L alpha to HII phase transition with a similar change in motional averaging as observed for the phospholipid chains themselves. In contrast, decane is apparently deeply embedded in the lipid structure and exhibits only a small degree of orientation. The L alpha to HII phase transition for systems containing decane leads to a dramatic increase of the motional freedom of decane which is more pronounced than that observed for the lipid chains. This is consistent with a preferential partition of the decane molecules into a disordered environment such as the intercylinder spaces in the HII phase. The presence of decane in the HII phase structure does not modify the order of the lipid chains.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of glycerol on the phase behaviour of hydrated distearoylphosphatidylethanolamine and its possible relation to the mode of action of cryoprotectants

A phase diagram for 1,2-distearoylphosphatidylethanolamine (DSPE) dispersed in glycerol/water mixtures was constructed using data obtained from differential scanning calorimetry and time-resolved X-ray diffraction measurements. The phase sequence seen on heating the lipid remains the same for samples containing up to 70 wt% glycerol. Depending on the hydration conditions, the samples are either in a metastable lamellar gel (L beta) or one or other of two possible sub-gel phases (Lc and Lc') at low temperatures. These phases convert first to a lamellar liquid crystalline (L alpha) and then to an inverted hexagonal (HII) phase on heating. On cooling, the samples revert first to the L alpha and then to the L beta phase. Although the phase sequence is preserved, marked changes are seen in the transition temperatures between the different phases. The temperature of the transition between the L alpha and the HII phases decreases strongly with increasing glycerol concentration while that of the Lc and Lc' phases to L alpha, and to a lesser extent that of the L beta to L alpha transition, increases. Substantial changes in phase behaviour are seen if the glycerol concentration is increased above 70 wt%. Under these conditions, the Lc and Lc' phases transform directly into the HII phase on heating (a similar direct transition from the L beta to the HII phase is seen above 80 wt% glycerol). An exothermic transition from the L beta phase to the Lc' phase is observed and there is also an increasing tendency for the samples to revert to the Lc or Lc' phases on storage. These changes in relative stability of the different phases are discussed in terms of a possible membrane Hofmeister effect and their relevance to the mode of action of cryoprotectants is explored.     

Chemical exchange between lamellar and non-lamellar lipid phases. A one- and two-dimensional 31P-NMR study.

One- and two-dimensional 31P-exchange NMR has been used to investigate chemical exchange between coexisting lamellar (L alpha) and non-lamellar (hexagonal HII and cubic I2) lipid phases. Samples of DOPE, DOPE/DOPC (9:1 and 7:3), DOPE/cholesterol sulfate (9:1), DOPC/monoolein (MO) (3:7 and 1:1), and DOPC/DOPE/cholesterol (1:1:2) were macroscopically oriented on glass plates and studied at the 0 degree orientation (angle between the bilayer normal and the external magnetic field), where the L alpha, HII, and I2 resonances are resolved. A reversible L alpha to HII transition was observed for all of the samples except for the DOPC/MO mixtures, which displayed a reversible L alpha to I2 transition. Near-equilibrium mixtures of L alpha and either HII or I2 were obtained after prolonged incubation at a given temperature. Two-dimensional exchange experiments were performed on DOPE at 9-14 degrees C for mixing times ranging from 500 ms to 2 s. For all samples, one-dimensional exchange experiments were performed for mixing times ranging from 100 ms to 4 s, at temperatures ranging from 3 degrees C to 73 degrees C. No evidence of lipid exchange between lamellar and non-lamellar phases was observed, indicating that if such a process occurs it is either very slow on the seconds' timescale, or involves an undetectable quantity of lipid. The results place constraints on the stability or kinetic behaviour of proposed transition intermediates (Siegel, D.P. (1986) Biophys. J. 49, 1155-1170).     

Phase equilibria of membrane lipids from Acholeplasma laidlawii: importance of a single lipid forming nonlamellar phases

A basis for the reorganization of the bilayer structure in biological membranes is the different aggregate structures formed by lipids in water. The phase equilibria of all individual lipids and several in vivo polar lipid mixtures from acyl chain modified membranes of Acholeplasma laidlawii were investigated with different NMR techniques. All dioleoyl (DO) polar lipids, except monoglucosyldiglyceride (MGDG), form lamellar liquid crystalline (L alpha) phases only. The phase diagram of DOMGDG reveals reversed cubic (III), reversed hexagonal (HII), and L alpha phases. In mixtures of DOMGDG and dioleoyldiglycosyldiglyceride (DODGDG), the formation of an III (or HII) phase is enhanced by DOMGDG and low hydration or high temperatures. For in vivo mixtures of all polar DO lipids, a transition from an L alpha to an III phase is promoted by low hydration or high temperatures (50 degrees C). The phospholipids are incorporated in this III phase. Likewise, III and HII phases are formed at similar temperatures in a series of in vivo mixtures with different extents of acyl chain unsaturation. However, their melting temperatures (Tm) vary in an expected manner. All cubic and hexagonal phases, except the III phase with DOMGDG, exist in equilibrium with excess water. The maximum hydration of MGDG and DGDG is similar and increases with acyl chain unsaturation but is substantially lower than that for, e.g., phosphatidylcholine. The translational diffusion of the lipids in the cubic phases is rapid, implying bicontinuous structures. However, their appearances in freeze-fracture electron microscope pictures are different. The III phase of DOMGDG belongs to the Ia3d space group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and interactions of ether- and ester-linked phosphatidylethanolamines

The ether-linked phospholipid 1,2-dihexadecylphosphatidylethanolamine (DHPE) was studied as a function of hydration and in fully hydrated mixed phospholipid systems with its ester-linked analogue 1,2-dipalmitoylphosphatidylethanolamine (DPPE). A combination of differential scanning calorimetry (DSC) and X-ray diffraction was used to examine the phase behavior of these lipids. By DSC, from 0 to 10 wt % H2O, DHPE displayed a single reversible transition that decreased from 95.2 to 78.8 degrees C and which was shown by X-ray diffraction data to be a direct bilayer gel to inverted hexagonal conversion, L beta----HII. Above 15% H2O, two reversible transitions were observed which stabilized at 67.1 and 92.3 degrees C above 19% H2O. X-ray diffraction data of fully hydrated DHPE confirmed the lower temperature transition to be a bilayer gel to bilayer liquid-crystalline (L beta----L alpha) phase transition and the higher temperature transition to be a bilayer liquid-crystalline to inverted hexagonal (L alpha----HII) phase transition. The lamellar repeat distance of gel-state DHPE increased as a function of hydration to a limiting value of 62.5 A at 19% H2O (8.6 mol of water/mol of DHPE), which corresponds to the hydration at which the transition temperatures are seen to stabilize by DSC. Electron density profiles of DHPE, in addition to calculations of the lipid layer thickness, confirmed that DHPE in the gel state forms a noninterdigitated bilayer at all hydrations. Fully hydrated mixed phospholipid systems of DHPE and DPPE exhibited two reversible transitions by DSC.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)