Molecular view of hexagonal phase formation in phospholipid membranes

Important biological processes, such as vesicle fusion or budding, require the cell matrix to undergo a transition from a lamellar to a nonlamellar state. Although equilibrium properties of membranes are amenable to detailed theoretical studies, collective rearrangements involved in phase transitions have thus far only been modeled on a qualitative level. Here, for the first time, the complete transition pathway from a multilamellar to an inverted hexagonal phase is elucidated at near-atomic detail using a recently developed coarse-grained molecular dynamics simulation model. Insight is provided into experimentally inaccessible data such as the molecular structure of the intermediates and the kinetics involved. Starting from multilamellar configurations, the spontaneous formation of stalks between the bilayers is observed on a nanosecond timescale at elevated temperatures or reduced hydration levels. The stalks subsequently elongate in a cooperative manner leading to the formation of an inverted hexagonal phase. The rate of stalk elongation is approximately 0.1 nm ns(-1). Within a narrow hydration/temperature/composition range the stalks appear stable and rearrange into the rhombohedral phase.     

Destabilization of phosphatidylethanolamine liposomes at the hexagonal phase transition temperature

We have examined whether there is a relationship between the lamellar-hexagonal phase transition temperature, TH, and the initial kinetics of H+- and Ca2+-induced destabilization of phosphatidylethanolamine (PE) liposomes. The liposomes were composed of dioleoylphosphatidylethanolamine, egg phosphatidylethanolamine (EPE), or phosphatidylethanolamine prepared from egg phosphatidylcholine by transesterification (TPE). These lipids have well-spaced lamellar-hexagonal phase transition temperatures (approximately 12, approximately 45, and approximately 57 degrees C) in a temperature range that allows us to measure the initial kinetics of bilayer destabilization, both below and above TH. The liposomes were prepared at pH 9.5. The TH of EPE and TPE was measured by using differential scanning calorimetry, and it was found that the TH was essentially the same at low pH or at high pH in the presence of 20 mM Ca2+. At temperatures well below TH, either at pH 4.5 or at pH 9.5 in the presence of Ca2+, the liposomes aggregate, leak, and undergo lipid mixing and mixing of contents. We show that liposome/liposome contact is involved in the destabilization of the PE liposomes. The temperature dependence of leakage, lipid mixing, and mixing of contents shows that there is a massive enhancement in the rate of leakage when the temperature approaches the TH of the particular PE and that lipid mixing appears to be enhanced. However, the fusion (mixing of aqueous contents) is diminished or even abolished at temperatures above TH. At and above the TH, a new mechanism of liposome destabilization arises, evidently dependent upon the ability of the PE molecules to adapt new morphological structures at these temperatures. We propose that this destabilization demarks the first step in the pathway to the eventual formation of the HII phase. Thus, the polymorphism accessible to PE is a powerful agent for membrane destabilization, but additional factors are required for fusion.     

Destabilization of phosphatidylethanolamine-containing liposomes: hexagonal phase and asymmetric membranes

We have measured the temperature of the L alpha-HII phase transition, TH, for several types of phosphatidylethanolamine (PE), their binary mixtures, and several PE/cholesteryl hemisuccinate (CHEMS) mixtures. We have shown for liposomes composed of pure PE and in mixtures with CHEMS that there is an aggregation-mediated destabilization which is greatly enhanced at and above TH. We now ask the question: How well can a dioleoylphosphatidylethanolamine/CHEMS liposome, for example, destabilize TPE (transesterified from egg phosphatidylcholine)/CHEMS liposome and vice versa? We use Ca2+ and H+ to induce aggregation and to provide different values of TH: the TH of the PE/CHEMS mixture is much lower at low pH than with Ca2+. We find that if the temperature is above the TH of one lipid mixture, e.g., A, and below the TH of the other lipid mixture, e.g., B, then the destabilization sequence [measured by the fluorescent 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylylenebis(pyridinium bromide) leakage assay] is AA greater than AB much greater than BB. That is, the bilayer of the lipid A (which on its own would end up in the HII phase) destabilizes itself better than it destabilizes the bilayer of lipid B (which on its own would remain in the L alpha phase). The BB contact is the least unstable. From these experiments, we conclude that the enhanced destabilization of membranes provided by the polymorphism accessible to these lipids above TH is effective even if only one of the apposed outer monolayers is HII phase competent. The surprising result is that if the temperature is above the TH of both lipid mixtures, then the destabilization sequence is AB greater than AA, BB. That is, the mixed bilayers are destabilized more by contact than either of the pure pairs. We believe that this is due to specific differences in the kinetics of aggregation or close approach of the membranes. Similar results were obtained with pure PE liposomes induced to aggregate by Ca2+ at pH 9.5. We also found that the kinetics of low-pH-induced leakage from PE/CHEMS liposomes were initially faster when the CHEMS on both sides of the bilayer is fully protonated. However, in a citrate buffer, which cannot cross intact membranes, the leakage was eventually faster. Flip-flop of the protonated CHEMS to the inner monolayer can explain this observation.     

Influence of basic polypeptides on the phase transition of phospholipid liposomes

Interaction of basic polypeptides (copolymers of lysine with phenylalanine or tyrosine) with phosphatidyserine or dipalmitoyl phosphatidylcholine liposomes was investigated by differential scanning calorimetry. The polypeptides cause a decrease in enthalpy of melting of the phospholipids almost without affecting the midpoint melting temperature.     

Virus replication inhibitory peptide inhibits the conversion of phospholipid bilayers to the hexagonal phase

Virus replication inhibitory peptide (carbobenzoxy-D-Phe-L-PheGly) was shown to be a potent specific inhibitor of the replication of paramyxovirus and myxovirus (Richardson, Scheid and Choppin (1980), Virology105, 205–222). This peptide inhibits the membrane fusing activity of a viral glycoprotein.Many agents which promote the formation of the hexagonal phase in membranes also accelerate membrane fusion. At a mole fraction of 0.1, viral replication inhibitory peptide can raise the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine by almost 10°. Two related peptides, carbobenzoxy-L-PheGly and carbobenzoxy-L-GlyPhe, are less potent in raising the bilayer to hexagonal phase transition temperature, with the latter peptide being the least effective of the three. This order of potency is the same as the order of potency in inhibiting viral replication. Substances which inhibit hexagonal phase formation of pure lipids may also inhibit membrane fusion.Abbreviations DEPE dielaidoylphosphatidyethanolamine - Z carbobenzoxy - DSC differential scanning calorimetry - VRIP virus replication inhibitory peptide (Z-D-Phe-L-PheGly)     

External addition of gramicidin induces the hexagonal HII phase in dioleoylphosphatidylcholine model membranes

³¹P-NMR, small angle X-ray diffraction and freeze-fracture electron microscopy show that dioleoylphosphatidylcholine liposomes undergo a transition from the lamellar to the hexagonal H_II phase upon injection of an ethanolic solution of gramicidin in the aqueous medium, when the molar ratio of peptide to lipid is 1 to 20 or higher.     

The lamellar to hexagonal phase transition in phosphatidylethanolamine liposomes: a fluorescence anisotropy study

The steady-state anisotropy of trimethylammonium diphenylhexatriene fluorescence has been used to monitor the thermotropic lamellar to HII hexagonal phase transition in an unsaturated phosphatidylethanolamine. The transition is observed in lipid aggregates when they are heated above the transition temperature Th, as well as in diluted liposomes after aggregation above Th. Changes in fluorescence anisotropy are not observed with Ca(++)-induced fusion of phosphatidylserine vesicles, a process not involving hexagonal phase formation.     

Myelin basic protein induces hexagonal phase formation in dispersions of diacylphosphatidic acid

31P nuclear magnetic resonance and low-angle X-ray diffraction measurements have shown that the basic protein of myelin caused diacylphosphatidic acid dispersions to change from a lamellar to a hexagonal lipid organisation. Several other basic proteins failed to effect a similar phase change, and had little influence on phospholipid headgroup structure and motion.     

Oxidized phosphatidylcholines facilitate phospholipid flip-flop in liposomes

Lipid asymmetry is a ubiquitous property of the lipid bilayers in cellular membranes and its maintenance and loss play important roles in cell physiology, such as blood coagulation and apoptosis. The resulting exposure of phosphatidylserine on the outer surface of the plasma membrane has been suggested to be caused by a specific membrane enzyme, scramblase, which catalyzes phospholipid flip-flop. Despite extensive research the role of scramblase(s) in apoptosis has remained elusive. Here, we show that phospholipid flip-flop is efficiently enhanced in liposomes by oxidatively modified phosphatidylcholines. A combination of fluorescence spectroscopy and molecular dynamics simulations reveal that the mechanistic basis for this property of oxidized phosphatidylcholines is due to major changes imposed by the oxidized phospholipids on the biophysical properties of lipid bilayers, resulting in a fast cross bilayer diffusion of membrane phospholipids and loss of lipid asymmetry, requiring no scramblase protein.     

Iron binding to alpha-tocopherol-containing phospholipid liposomes

Tocopherols (vitamin E) located in the hydrophobic domains of biological membranes act as chain breaking antioxidants preventing the propagation of free radical reactions of lipid peroxidation. The naturally occurring form, d-alpha tocopherol is an exquisite molecule in that it is intercalated in the membrane in such a way that the hydrophobic tail anchors the molecule positioning the chromanol ring containing the hydroxyl group, which is the essence of its antioxidant function, at the polar hydrocarbon interface of phospholipid membranes. The interaction of this group with water soluble substances is not very well understood. In the present study, an investigation was made of the interaction of ascorbate and ferrous ions (Fe+2) initiators of lipid peroxidation with alpha tocopherol. The results show that tocopherol increases membrane associated iron. The formation of a tocopherol iron complex in the presence of phospholipid liposomes and ascorbate in its reduced form is indicated. These results suggest a new way in which tocopherols act to inhibit lipid peroxidation.     

Interaction of isopropylthioxanthone with phospholipid liposomes

Isopropylthioxanthone (ITX) is a highly lipophilic molecule which can be released in foods and beverages from the packages, where it is present as photoinitiator of inks in printing processes. Recently it was found in babies milk, and its toxicity cannot be excluded. The structure of the molecule suggests a possible strong interaction with the lipid moiety of biological membranes, and this is the first study of its effects on phospholipid organization, using differential scanning calorimetry (DSC) and spin labelling techniques. The data obtained with multilamellar liposomes of saturated phospholipids of different length, with and without cholesterol, point out that the molecule changes the lipid structure; in particular, in the gel state, behaving like a disordering agent it increases the mobility of the bilayer, while, in the fluid state, tends to rigidify the membrane, in a cholesterol like way. This behavior supports the hypothesis that ITX experiences a relocation process when the lipid matrix passes from the gel to the fluid state.     

Radiation-induced changes in permeability in unilamellar phospholipid liposomes

Gamma-radiation-induced oxidative damage in unilamellar dipalmitoylphosphatidylcholine liposomes was investigated using a fluorescence technique. Liposomal changes in permeability induced by gamma radiation were monitored by measuring the leakage of pre-encapsulated 6-carboxyfluorescein, and alterations in lipid bilayer fluidity were determined by 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization. The changes in permeability and fluidity in the bilayer were found to be dependent on the radiation dose in a biphasic fashion. The results are interpreted in terms of lipid bilayer fluidization after exposure to doses up to 1 kGy, but rigidization of the bilayer at higher doses. These results indicate a relationship between alterations in permeability and fluidity in the lipid bilayer after irradiation. The vesicles were protected significantly against radiation-induced oxidative damage in the presence of alpha-tocopherol and ascorbic acid. Radiation-induced changes in the permeability of the liposomes after exposure to gamma radiation and their modification by antioxidants indicate the involvement of a free radical mechanism in the production of damage, which may offer new insights in to the modification of cellular radiosensitivity by modulation of membrane damage.     

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.     

Studies on lipid oxidation in fish phospholipid liposomes

Fish phospholipid liposomes were prepared and used as an artificial membrane system to study factors influencing-lipid oxidation. The extent of lipid oxidation was indexed by measuring the amount of thiobarbituric acid reactive substances (TBARS) produced. Fe²⁺, Fe³⁺, and Cu²⁺ were potent prooxidants in catalysing lipid oxidation. These metal ions induced lipid oxidation in a dose dependent manner. However, Zn²⁺, Ni²⁺, and Mn²⁺ did not significantly (p>0.05) affect lipid oxidation at all the concentrations (1, 10, or 100 μM) studied. Morin, luteolin (flavonoids), butein (chalcone), tannic acid, ellagic acid (polyphenols), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) (synthetic antioxidants) were potent antioxidants (producing <50% TBARS compared to control) of Fe²⁺-catalyzed lipid oxidation. Morin, luteolin, and butein possess two hydroxyl substituents, a C₄ ketone structure and a 2–3 double bond, all of which contributed to their antioxidative potential. Fe²⁺ caused some losses of polyunsaturated fatty acids (PUFA), whereas tannic acid protected the oxidation of several of the PUFA including C 16∶1 (Palmitoleic acid), C 18∶3 (Linolenic acid), C 20∶4 (Arachidonic acid), C 20∶5 (Eicosapentaenoic acid), and C 22∶6 (Docosahexaenoic acid).     

Relationship between gramicidin conformation dependent induction of phospholipid transbilayer movement and hexagonal HII phase formation in erythrocyte membranes

Addition of gramicidin in sufficient concentration from dimethylsulfoxide or trifluoroethanol to isolated erythrocyte membranes induces hexagonal HII phase formation for the phospholipids. In contrast, addition from ethanol does not change the overall bilayer organization despite a similar extent of peptide incorporation. The same solvent dependence is observed for the enhancement of transbilayer reorientation of lysophospholipids and unspecific leak formation in intact erythrocytes at lower gramicidin concentrations. These results indicate that the (beta 6.3) conformation of the peptide is essential for all three membrane perturbing effects.     

Hematin- and peroxide-catalyzed peroxidation of phospholipid liposomes

The effect of hydroperoxides on hematin-catalyzed initiation and propagation of lipid peroxidation was examined utilizing soybean phosphatidylcholine liposomes as model membranes. Polarographic and spectrophotometric methods revealed a bimodal pseudocatalytic activity for hematin. A slow initiation phase of peroxidation was observed in the presence of low peroxide concentrations, whereas a fast propagative phase was observed at higher peroxide levels. Peroxide levels were manipulated enzymatically by the combination of phospholipase A2 and lipoxidase or by the direct addition of linoleic acid hydroperoxide, cumene hydroperoxide, or hydrogen peroxide. In addition, the effect of two different techniques for liposome preparation, i.e., sonication and extrusion, were compared on the basis of peroxidation kinetics. High pressure liquid chromatography analysis showed that sonicated liposomes contained higher levels of endogenous peroxides than the extruded ones. These sonicated liposomes also exhibited more rapid peroxidation following hematin addition. Extruded liposomes were more resistant to hematin-catalyzed peroxidation but became better substrates when exogenous hydroperoxides were added. All three peroxides reacted with hematin during which decomposition of peroxide and irreversible oxidation of hematin took place. Spectral analysis of hematin indicated that a higher oxidation state of hematin iron may be transiently formed during reaction with hydroperoxides and accounts for the propagation of lipid peroxidation when reactions proceed in the presence of soybean phosphatidylcholine liposomes. Of the three peroxides studied, linoleic acid hydroperoxide was most efficient in supporting hematin-catalyzed lipid peroxidation. The relevance of our findings is discussed in terms of the concentration dependence for lipid peroxides in determining the rate and extent of radical propagation chain reactions catalyzed by heme-iron catalysts such as hematin. Variation of hematin and linoleic hydroperoxide concentrations may provide an efficient and reproducible method for inducing and manipulating the rates and extent of lipid peroxidation through facilitation of the propagative phase of lipid peroxidation. In addition, we address a problem inherent to in vitro studies of heme-catalyzed lipid peroxidation where preparations of peroxide-free membranes should be of concern.     

The measurement of malondialdehyde in peroxidised ox-brain phospholipid liposomes

The preparation of ox-brain phospholipid liposomes and their peroxidation using different catalysts have been described in detail. The degree of peroxidation is related to the formation of thiobarbituric acid (TBA)-reacting compounds and expressed as malondialdehyde (MDA). As confirmatory data, fluorescent MDA-phospholipid complexes were measured in parallel. Close agreement between the polar TBA-reactive compounds and the nonpolar fluorescent compounds confirmed the usefulness of the simple TBA test as a measure of peroxidative activity in pure lipid liposomes. Relative differences in the catalytic activity of ascorbic acid and cupric ions when assayed by the two methods are discussed.