Energetics of a hexagonal-lamellar-hexagonal-phase transition sequence in dioleoylphosphatidylethanolamine membranes.

The phase diagram of DOPE/water dispersions was investigated by NMR and X-ray diffraction in the water concentration range from 2 to 20 water molecules per lipid and in the temperature range from -5 to +50 degrees C. At temperatures above 22 degrees C, the dispersions form an inverse (HII) phase at all water concentrations. Below 25 degrees C, an HII phase occurs at high water concentrations, an L alpha phase is formed at intermediate water concentrations, and finally the system switches back to an HII phase at low water concentrations. The enthalpy of the L alpha-HII-phase transition is +0.3 kcal/mol as measured by differential scanning calorimetry. Using 31P and 2H NMR and X-ray diffraction, we measured the trapped water volumes in HII and L alpha phases as a function of osmotic pressure. The change of the HII-phase free energy as a function of hydration was calculated by integrating the osmotic pressure vs trapped water volume curve. The phase diagram calculated on the basis of the known enthalpy of transition and the osmotic pressure vs water volume curves is in good agreement with the measured one. The HII-L alpha-HII double-phase transition at temperatures below 22 degrees C can be shown to be a consequence of (i) the greater degree of hydration of the HII phase in excess water and (ii) the relative sensitivities with which the lamellar and hexagonal phases dehydrate with increasing osmotic pressure. These results demonstrate the usefulness of osmotic stress measurements to understand lipid-phase diagrams.     

Bending Rigidities and Interdomain Forces in Membranes with Coexisting Lipid Domains

To precisely quantify the fundamental interactions between heterogeneous lipid membranes with coexisting liquid-ordered (Lo) and liquid-disordered (Ld) domains, we performed detailed osmotic stress small-angle x-ray scattering experiments by exploiting the domain alignment in raft-mimicking lipid multibilayers. Performing a Monte Carlo-based analysis allowed us to determine with high reliability the magnitude and functional dependence of interdomain forces concurrently with the bending elasticity moduli. In contrast to previous methodologies, this approach enabled us to consider the entropic undulation repulsions on a fundamental level, without having to take recourse to crudely justified mean-field-like additivity assumptions. Our detailed Hamaker-coefficient calculations indicated only small differences in the van der Waals attractions of coexisting Lo and Ld phases. In contrast, the repulsive hydration and undulation interactions differed significantly, with the latter dominating the overall repulsions in the Ld phase. Thus, alignment of like domains in multibilayers appears to originate from both, hydration and undulation repulsions.     

Can hydration forces induce lateral phase separations in lamellar phases?

Large repulsive forces measured between membranes of lamellar lipid phases at low hydration are attributed to hydration interactions which vary widely among lipid species. We include this interaction in a model of lamellar phases of two membrane components (two lipids or lipid and protein). The surface polarization of a mixture is taken as a linear combination of those of the components. The model predicts phase separation at low hydration. This may have important consequences for living cells which are dehydrated either by the osmotic effects of tissue freezing, or by desiccation in unsaturated atmospheres.Abbreviations used ACC cold acclimated protoplasts - NA non cold acclimated protoplasts - DLPC dilauralphosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - DPPE dipalmitoylphosphatidylethanolamine - PC phosphatidylcholine - PE phosphatidylethanolamine - L fluid lamellar phase - H_ii inverse hexagonal phase     

Osmotically Induced Reversible Transitions in Lipid-DNA Mesophases

We follow the effect of osmotic pressure on isoelectric complexes that self-assemble from mixtures of DNA and mixed neutral and cationic lipids. Using small angle x-ray diffraction and freeze-fracture cryo-electron microscopy, we find that lamellar complexes known to form in aqueous solutions can reversibly transition to hexagonal mesophases under high enough osmotic stress exerted by adding a neutral polymer. Using molecular spacings derived from x-ray diffraction, we estimate the reversible osmotic pressure-volume (Π-V) work needed to induce this transition. We find that the transition free energy is comparable to the work required to elastically bend lipid layers around DNA. Consistent with this, the required work is significantly lowered by an addition of hexanol, which is known to soften lipid bilayers. Our findings not only help to resolve the free-energy contributions associated with lipid-DNA complex formation, but they also demonstrate the importance that osmotic stress can have to the macromolecular phase geometry in realistic biological environments.     

The phase behavior of cationic lipid-DNA complexes

We present a theoretical analysis of the phase behavior of solutions containing DNA, cationic lipids, and nonionic (helper) lipids. Our model allows for five possible structures, treated as incompressible macroscopic phases: two lipid-DNA composite (lipoplex) phases, namely, the lamellar (L(alpha)(C)) and hexagonal (H(II)(C)) complexes; two binary (cationic/neutral) lipid phases, that is, the bilayer (L(alpha)) and inverse-hexagonal (H(II)) structures, and uncomplexed DNA. The free energy of the four lipid-containing phases is expressed as a sum of composition-dependent electrostatic, elastic, and mixing terms. The electrostatic free energies of all phases are calculated based on Poisson-Boltzmann theory. The phase diagram of the system is evaluated by minimizing the total free energy of the three-component mixture with respect to all the compositional degrees of freedom. We show that the phase behavior, in particular the preferred lipid-DNA complex geometry, is governed by a subtle interplay between the electrostatic, elastic, and mixing terms, which depend, in turn, on the lipid composition and lipid/DNA ratio. Detailed calculations are presented for three prototypical systems, exhibiting markedly different phase behaviors. The simplest mixture corresponds to a rigid planar membrane as the lipid source, in which case, only lamellar complexes appear in solution. When the membranes are "soft" (i.e., low bending modulus) the system exhibits the formation of both lamellar and hexagonal complexes, sometimes coexisting with each other, and with pure lipid or DNA phases. The last system corresponds to a lipid mixture involving helper lipids with strong propensity toward the inverse-hexagonal phase. Here, again, the phase diagram is rather complex, revealing a multitude of phase transitions and coexistences. Lamellar and hexagonal complexes appear, sometimes together, in different regions of the phase diagram.     

Temperature and compositional dependence of the structure of hydrated dimyristoyl lecithin.

Differential scanning calorimetry and x-ray diffraction techniques have been used to investigate the structure and phase behavior of hydrated dimyristoyl lecithin (DML) in the hydration range 7.5 to 60 weight % water and the temperature range -10 to +60 degrees C. Four different calorimetric transitions have been observed: T1, a low enthalpy transition (deltaH approximately equal to 1 kcal/mol of DML) at 0 degrees C between lamellar phases (L leads to Lbeta); T2, the low enthalpy "pretransition" at water contents greater than 20 weight % corresponding to the transition Lbeta leads to Pbeta; T3, the hydrocarbon chain order-disorder transition (deltaH = 6 to 7 kcal/mol of DML) representing the transition of the more ordered low temperature phases (Lbeta, Pbeta, or crystal C, depending on the water content) to the lamellar Lalpha phase; T4, a transition occurring at 25--27 degrees C at low water contents representing the transition from the lamellar Lbeta phase to a hydrated crystalline phase C. The structures of the Lbeta, Pbeta, C, and Lalpha phases have been examined as a function of temperature and water content. The Lbeta structure has a lamellar bilayer organization with the hydrocarbon chains fully extended and tilted with respect to the normal to the bilayer plane, but packed in a distorted quasihexagonal lattice. The Pbeta structure consists of lipid bilayer lamellae distorted by a periodic "ripple" in the plane of the lamellae; the hydrocarbon chains are tilted but appear to be packed in a regular hexagonal lattice. The diffraction pattern from the crystalline phase C indexes according to an orthorhombic cell with a = 53.8 A, b = 9.33 A, c = 8.82 A. In the lamellae bilayer Lalpha strucure, the hydrocarbon chains adopt a liquid-like conformation. Analysis of the hydration characteristics and bilayer parameters (lipid thickness, surface area/molecule) of synthetic lecithins permits an evaluation of the generalized hydration and structural behavior of this class of lipids.     

The inverse hexagonal - inverse ribbon - lamellar gel phase transition sequence in low hydration DOPC:DOPE phospholipid mixtures

The inverse hexagonal to inverse ribbon phase transition in a mixed phosphatidylcholine-phosphatidylethanolamine system at low hydration is studied using small and wide angle X-ray scattering. It is found that the structural parameters of the inverse hexagonal phase are independent of temperature. By contrast the length of each ribbon of the inverse ribbon phase increases continuously with decreasing temperature over a range of 50 °C. At low temperatures the inverse ribbon phase is observed to have a transition to a gel lamellar phase, with no intermediate fluid lamellar phase. This phase transition is confirmed by differential scanning calorimetry.     

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.     

New phases of phospholipids and implications to the membrane fusion problem

Membrane fusion is a ubiquitous process in eukaryotic cells. When two membranes fuse, lipid must undergo molecular rearrangements at the point of merging. To understand how lipid structure transitions occur, scientists studied the phase transition of lipid between the lamellar (L(alpha)) phase and the inverted hexagonal (H(II)) phase, based on the idea that lipid must undergo a similar rearrangement as in fusion. However, previous investigations on the system of dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE) did not reveal intermediate phases between the L(alpha) and H(II) phases. Recently, we found a rhombohedral phase of diphytanoylphosphatidylcholine between its L(alpha) and H(II) phases using substrate-supported samples. Here we report the observation of two new phases in the DOPC-DOPE system: a rhombohedral phase and a distorted hexagonal phase. The rhombohedral phase confirms the stalk hypothesis for the L(alpha)-H(II) transition, but the phase of stable stalks exists only for a certain range of spontaneous curvature. The distorted hexagonal phase exists only in a lipid mixture. It implies that lipids may demix to adjust its local spontaneous curvature in order to achieve energy minimum under stress.     

Theory of lipid polymorphism: application to phosphatidylethanolamine and phosphatidylserine

We introduce a microscopic model of a lipid with a charged headgroup and flexible hydrophobic tails, a neutral solvent, and counter ions. Short-ranged interactions between hydrophilic and hydrophobic moieties are included as are the Coulomb interactions between charges. Further, we include a short-ranged interaction between charges and neutral solvent, which mimics the short-ranged, thermally averaged interaction between charges and water dipoles. We show that the model of the uncharged lipid displays the usual lyotropic phases as a function of the relative volume fraction of the headgroup. Choosing model parameters appropriate to dioleoylphosphatidylethanolamine in water, we obtain phase behavior that agrees well with experiment. Finally we choose a solvent concentration and temperature at which the uncharged lipid exhibits an inverted hexagonal phase and turn on the headgroup charge. The lipid system makes a transition from the inverted hexagonal to the lamellar phase, which is related to the increased waters of hydration correlated with the increased headgroup charge via the charge-solvent interaction. The polymorphism displayed upon variation of pH mimics that of the behavior of phosphatidylserine.     

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.     

The effects of solutes on the freezing properties of and hydration forces in lipid lamellar phases.

Quantitative deuterium nuclear magnetic resonance is used to study the freezing behavior of the water in phosphatidylcholine lamellar phases, and the effect upon it of dimethylsulfoxide (DMSO), sorbitol, sucrose, and trehalose. When sufficient solute is present, an isotropic phase of concentrated aqueous solution may coexist with the lamellar phase at freezing temperatures. We determine the composition of both unfrozen phases as a function of temperature by using the intensity of the calibrated free induction decay signal (FID). The presence of DMSO or sorbitol increases the hydration of the lamellar phase at all freezing temperatures studied, and the size of the increase in hydration is comparable to that expected from their purely osmotic effect. Sucrose and trehalose increase the hydration of the lamellar phase, but, at concentrations of several molal, the increase is less than that which their purely osmotic effect would be expected to produce. A possible explanation is that very high volume fractions of sucrose and trehalose disrupt the water structure and thus reduce the repulsive hydration interaction between membranes. Because of their osmotic effect, all of the solutes studied reduced the intramembrane mechanical stresses produced in lamellar phases by freezing. Sucrose and trehalose at high concentrations produce a greater reduction than do the other solutes.     

Phase behavior and structure of aqueous dispersions of sphingomyelin

The phase behavior of bovine brain sphingomyelin in water has been determined by polarizing light microscopy, differential scanning calorimetry, and X-ray diffraction. Lamellar phases, in which water is intercalated between sheets of lipid molecules arranged in the classical bilayer fashion, are present over much of the phase diagram. An order-disorder transition separates the high temperature, liquid crystalline, lamellar phase from a more ordered lamellar phase at low temperatures. The hydration characteristics of sphingomyelin are similar to the structurally related lecithin in that only limited amounts of water are incorporated above and below the transition. Above the transition at 47 degrees C, a maximum of 35% by weight of water can be incorporated between the lipid bilayers, the total thickness at maximum hydration being 60.2 A, the lipid thickness 38 A, and the surface area per lipid molecule at the interface 60 A(2). Water in excess of 35% by weight is present as a separate phase. Below the phase transition, at 25 degrees C a maximum of 42% by weight of water may be incorporated between the lipid bilayers. On increasing the hydration, the lamellar repeat distance increases from 63.5 A to a limiting value of 76 A. Within this hydration range the calculated lipid thickness decreases from 63.5 to 42.5 A, and the surface area per lipid molecule increases from 36.1 to 53.6 A(2). Although these changes may be accounted for by a structure in which the hexagonally packed ordered hydrocarbon chains tilt progressively with respect to the normal to the bilayer plane on increasing hydration, it is possible that changes in other more complex lamellar structures may be responsible for these variations in lipid thickness and surface area.     

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).     

Effect of fatty acyl chain length and structure on the lamellar gel to liquid-crystalline and lamellar to reversed hexagonal phase transitions of aqueous phosphatidylethanolamine dispersions

The lamellar gel/liquid-crystalline and the lamellar liquid-crystalline/reversed hexagonal phase transitions of aqueous dispersions of a number of synthetic phosphatidylethanolamines containing linear saturated, branched chain, and alicyclic fatty acyl chains of varying length were studied by differential scanning calorimetry, 31P nuclear magnetic resonance spectroscopy, and X-ray diffraction. For any given homologous series of phosphatidylethanolamines containing a single chemical class of fatty acids, the lamellar gel/liquid-crystalline phase transition temperature increases and the lamellar liquid-crystalline/reversed hexagonal phase transition temperature decreases with increases in hydrocarbon chain length. For a series of phosphatidylethanolamines of the same hydrocarbon chain length but with different chemical structures, both the lamellar gel/liquid-crystalline and the lamellar liquid-crystalline/reversed hexagonal phase transition temperatures vary markedly and in the same direction. In particular, at comparable effective hydrocarbon chain lengths, both the lamellar gel/liquid-crystalline and the lamellar liquid-crystalline/reversed hexagonal phase transition temperatures vary in parallel, such that the temperature difference between these two phase transitions is nearly constant. Moreover, at comparable effective acyl chain lengths, the d spacings of the lamellar liquid-crystalline phases and of the inverted hexagonal phases are all similar, implying that the thickness of the phosphatidylethanolamine bilayers at the onset of the lamellar liquid-crystalline/reversed hexagonal phase transition and the diameter of the water-filled cylinders formed at the completion of this phase transition are comparable and independent of the chemical structure of the acyl chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Order and dynamics in the lamellar L alpha and in the hexagonal HII phase. Dioleoylphosphatidylethanolamine studied with angle-resolved fluorescence depolarization.

Fluorescence depolarization techniques are used to determine the molecular order and reorientational dynamics of the probe molecule TMA-DPH embedded in the lamellar L alpha and the hexagonal HII phases of lipid/water mixtures. The thermotropically induced L alpha----HII phase transition of the lipid DOPE is used to obtain macroscopically aligned samples in the hexagonal HII phase at 45 degrees C from samples prepared in the lamellar L alpha phase at 7 degrees C. The interpretation of angle-resolved fluorescence depolarization experiments on these phases, within the framework of the rotational diffusion model, yields the order parameters (P2) and (P4), and the diffusion constants for the reorientational motions. The reorientational motion rates of the TMA-DPH molecules in the hexagonal HII phase are comparable with those in the lamellar L alpha phase. Furthermore, the lateral diffusion of the probe molecule on the surface of the lipid/water cylinder in the hexagonal phase is found to be considerably slower than the reorientational motion.     

Is an orthorhombic lateral packing and a proper lamellar organization important for the skin barrier function?

The lipid organization in the stratum corneum (SC), plays an important role in the barrier function of the skin. SC lipids form two lamellar phases with a predominantly orthorhombic packing. In previous publications a lipid model was presented, referred to as the stratum corneum substitute (SCS), that closely mimics the SC lipid organization and barrier function. Therefore, the SCS serves as a unique tool to relate lipid organization with barrier function. In the present study we examined the effect of the orthorhombic to hexagonal phase transition on the barrier function of human SC and SCS. In addition, the SCS was modified by changing the free fatty acid composition, resulting in a hexagonal packing and perturbed lamellar organization. By measuring the permeability to benzoic acid as function of temperature, Arrhenius plots were constructed from which activation energies were calculated. The results suggest that the change from orthorhombic to hexagonal packing in human SC and SCS, does not have an effect on the permeability. However, the modified SCS revealed an increased permeability to benzoic acid, which we related to its perturbed lamellar organization. Thus, a proper lamellar organization is more crucial for a competent barrier function than the presence of an orthorhombic lateral packing.     

Measurement and modification of forces between lecithin bilayers.

We probe in two different ways the competing attractive and repulsive forces that create lamellar arrays of the phospholipid lecithin when in equilibrium with pure water. The first probe involves the addition of low molecular weight solutes, glucose and sucrose, to a system where the phospholipid is immersed in a large excess of water. Small solutes can enter the aqueous region between bilayers. Their effect is first to increase and then to decrease the separation between bilayers as sugar concentration increases. We interpret this waxing and waning of the lattice spacing in terms of the successive weakening and strengthening of the attractive van der Waals forces originally responsible for creation of a stable lattice. The second probe is an "osmotic stress method," in which very high molecular weight neutral polymer is added to the pure water phase but is unable to enter the multilayers. The polymer competes for water with the lamellar lattice, and thereby compresses it. From the resulting spacing (determined by X-ray diffraction) and the directly measured osmotic pressure, we find a force vs. distance curve for compressing the lattice (or, equivalently, the free energy of transfer to bulk water of water between bilayers. This method reveals a very strong, exponentially varying "hydration force" with a decay distance of about 2 A.     

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.