Formation Of Nonlamellar Phases Research Articles

Coronavirus-Induced Host Cubic Membranes and Lipid-Related Antiviral Therapies: A Focus on Bioactive Plasmalogens.

Coronaviruses have lipid envelopes required for their activity. The fact that coronavirus infection provokes the formation of cubic membranes (CM) (denoted also as convoluted membranes) in host cells has not been rationalized in the development of antiviral therapies yet. In this context, the role of bioactive plasmalogens (vinyl ether glycerophospholipids) is not completely understood. These lipid species display a propensity for non-lamellar phase formation, facilitating membrane fusion, and modulate the activity of membrane-bound proteins such as enzymes and receptors. At the organism level, plasmalogen deficiency is associated with cardiometabolic disorders including obesity and type 2 diabetes in humans. A straight link is perceived with the susceptibility of such patients to SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2) infection, the severity of illness, and the related difficulty in treatment. Based on correlations between the coronavirus-induced modifications of lipid metabolism in host cells, plasmalogen deficiency in the lung surfactant of COVID-19 patients, and the alterations of lipid membrane structural organization and composition including the induction of CM, we emphasize the key role of plasmalogens in the coronavirus (SARS-CoV-2, SARS-CoV, or MERS-CoV) entry and replication in host cells. Considering that plasmalogen-enriched lung surfactant formulations may improve the respiratory process in severe infected individuals, plasmalogens can be suggested as an anti-viral prophylactic, a lipid biomarker in SARS-CoV and SARS-CoV-2 infections, and a potential anti-viral therapeutic component of lung surfactant development for COVID-19 patients.

An Evidence for a Novel Antiviral Mechanism: Modulating Effects of Arg-Glc Maillard Reaction Products on the Phase Transition of Multilamellar Vesicles.

Maillard reaction products (MRPs) of protein, amino acids, and reducing sugars from many foods and aqueous extracts of herbs are found to have various bioactivities, including antiviral effects. A hypothesis was proposed that their antiviral activity is due to the interaction with the cellular membrane. Aiming to estimate the possible actions of MRPs on phospholipid bilayers, the Arg-Glc MRPs were prepared by boiling the pre-mixed solution of arginine and glucose for 60 min at 100°C and then examined at a series of concentrations for their effects on the phase transition of MeDOPE multilamellar vesicles (MLVs), for the first time, by using differential scanning calorimetry (DSC) and temperature-resolved small-angle X-ray scattering (SAXS). Arg-Glc MRPs inhibited the lamellar gel–liquid crystal (Lβ-Lα), lamellar liquid crystal–cubic (Lα-QII), and lamellar liquid crystal–inverted hexagonal (Lα-HII) phase transitions at low concentration (molar ratio of lipid vs. MRPs was 100:1 or 100:2), but promoted all three transitions at medium concentration (100:5). At high concentration (10:1), the MRPs exhibited inhibitory effect again. The fusion peptide from simian immunodeficiency virus (SIV) induces membrane fusion by promoting the formation of a non-lamellar phase, e.g., cubic (QII) phase, and inhibiting the transition to HII. Arg-Glc MRPs, at low concentration, stabilized the lamellar structure of SIV peptide containing lipid bilayers, but facilitated the formation of non-lamellar phases at medium concentration (100:5). The concentration-dependent activity of MRPs upon lipid phase transition indiciates a potential role in modulating some membrane-related biological events, e.g., viral membrane fusion.

The plant dehydrin Lti30 stabilizes lipid lamellar structures in varying hydration conditions

A major challenge to plant growth and survival are changes in temperature and diminishing water supply. During acute temperature and water stress, plants often express stress proteins, such as dehydrins, which are intrinsically disordered hydrophilic proteins. In this article, we investigated how the dehydrin Lti30 from Arabidopsis thaliana stabilizes membrane systems that are exposed to large changes in hydration. We also compared the effects of Lti30 on membranes with those of the simple osmolytes urea and trimethylamine N-oxide. Using X-ray diffraction and solid-state NMR, we studied lipid-protein self-assembly at varying hydration levels. We made the following observations: 1) the association of Lti30 with anionic membranes relies on electrostatic attraction, and the protein is located in the bilayer interfacial membrane region; 2) Lti30 can stabilize the lamellar multilayer structure, making it insensitive to variations in water content; 3) in lipid systems with a composition similar to those present in some seeds and plants, dehydrin can prevent the formation of nonlamellar phases upon drying, which may be crucial for maintaining membrane integrity; and 4) Lti30 stabilizes bilayer structures both at high and low water contents, whereas the small osmolyte molecules mainly prevent dehydration-induced transitions. These results corroborate the idea that dehydrins are part of a sensitive and multifaceted regulatory mechanism that protects plant cells against stress.

Toward the Rational Design of Lipid Gene Vectors: Shape Coupling between Lipoplex and Anionic Cellular Lipids Controls the Phase Evolution of Lipoplexes and the Efficiency of DNA Release

A viewpoint now emerging is that a critical factor in lipid-mediated transfection (lipofection) is the structural evolution of lipoplexes upon interaction with anionic cellular lipids, resulting in DNA release. At the early stages of interaction, we found a universal behavior of lipoplex/anionic lipid (AL) mixtures: the lipoplex structure is slightly perturbed, while the one-dimensional DNA lattice between cationic membranes is largely diluted by ALs. This finding is in excellent agreement with previous suggestions on the mechanism of DNA unbinding from lipoplexes by ALs. Upon further interaction, the propensity of a given lipoplex structure to be solubilized by anionic cellular lipids strongly depends on the shape coupling between lipoplex and ALs. Furthermore, we investigated the effect of the membrane charge density and a general correlation resulted: the higher the membrane charge density of anionic membranes, the higher their ability to solubilize the structure of lipoplexes and to promote DNA release. Lastly, the formation of nonlamellar phases in lipoplex/AL mixtures is regulated by the propensity of anionic cellular lipids to adopt nonlamellar phases. Remarkably, also phase transition rates and DNA release were found to be strongly affected by the shape coupling between lipoplex and ALs. It thus seems likely that the structural and phase evolution of lipoplexes may only be meaningful in the context of specific anionic cellular membranes. These results highlight the phase properties of the carrier lipid/cellular lipid mixtures as a decisive factor for optimal DNA release and suggest a potential strategy for the rational design of efficient cationic lipid carriers.

Modulation of a membrane lipid lamellar–nonlamellar phase transition by cationic lipids: A measure for transfection efficiency

Synthetic cationic lipids can be used as DNA carriers and are regarded to be the most promising non-viral gene carriers. For this investigation, six novel phosphatidylcholine (PC) cationic derivatives with various hydrophobic moieties were synthesized and their transfection efficiencies for human umbilical artery endothelial cells (HUAEC) were determined. Three compounds with relatively short, myristoleoyl or myristelaidoyl 14:1 chains exhibited very high activity, exceeding by ∼ 10 times that of the reference cationic derivative dioleoyl ethylPC (EDOPC). Noteworthy, cationic lipids with 14:1 hydrocarbon chains have not been tested as DNA carriers in transfection assays previously. The other three lipids, which contained oleoyl 18:1 and longer chains, exhibited moderate to weak transfection activity. Transfection efficiency was found to correlate strongly with the effect of the cationic lipids on the lamellar-to-inverted hexagonal, L α → H II, phase conversion in dipalmitoleoyl phosphatidylethanolamine dispersions (DPoPE). X-ray diffraction on binary DPoPE/cationic lipid mixtures showed that the superior transfection agents eliminated the direct L α → H II phase transition and promoted formation of an inverted cubic phase between the L α and H II phases. In contrast, moderate and weak transfection agents retained the direct L α → H II transition but shifted to higher temperatures than that of pure DPoPE, and induced cubic phase formation at a later stage. On the basis of current models of lipid membrane fusion, promotion of a cubic phase by the high-efficiency agents may be considered as an indication that their high transfection activity results from enhanced lipoplex fusion with cellular membranes. The distinct, well-expressed correlation established between transfection efficiency of a cationic lipid and the way it modulates nonlamellar phase formation of a membrane lipid could be useful as a criterion to assess the quality of lipid carriers and for rational design of new and superior nucleotide delivery agents.

Membrane curvature stress and antibacterial activity of lactoferricin derivatives

We have studied correlation of non-lamellar phase formation and antimicrobial activity of two cationic amphipathic peptides, termed VS1-13 and VS1-24 derived from a fragment (LF11) of human lactoferricin on Escherichia coli total lipid extracts. Compared to LF11, VS1-13 exhibits minor, but VS1-24 significantly higher antimicrobial activity. X-ray experiments demonstrated that only VS1-24 decreased the onset of cubic phase formation of dispersions of E. coli lipid extracts, significantly, down to physiological relevant temperatures. Cubic structures were identified to belong to the space groups Pn3m and Im3m. Formation of latter is enhanced in the presence of VS1-24. Additionally, the presence of this peptide caused membrane thinning in the fluid phase, which may promote cubic phase formation. VS1-24 containing a larger hydrophobic volume at the N-terminus than its less active counterpart VS1-13 seems to increase curvature stress in the bilayer and alter the behaviour of the membrane significantly enhancing disruption.

On the correlation between phase evolution of lipoplexes/anionic lipid mixtures and DNA release

We investigated the structural evolution of three lipoplex formulations when interacting with anionic lipids by synchrotron small angle x-ray diffraction, while the extent of DNA release from lipoplexes by anionic lipids was evaluated by gel electrophoresis. Lipoplexes formed lamellar phases when mixed with anionic dioleoylphosphatidylglycerol (DOPG), while promoting the formation of nonbilayer structures when mixed with anionic dioleoylphosphatidic acid (DOPA). However, lipoplexes exhibited a virtually identical extent of DNA release when mixed with DOPG or DOPA. Thus, the recently proposed correlation between the formation of nonlamellar phases in lipoplex/anionic lipid mixtures and the increase of DNA release does not seem to exist.

An intracellular lamellar-nonlamellar phase transition rationalizes the superior performance of some cationic lipid transfection agents.

Two cationic phospholipid derivatives with asymmetric hydrocarbon chains were synthesized: ethyl esters of oleoyldecanoyl-ethylphosphatidylcholine (C18:1/C10-EPC) and stearoyldecanoyl-ethylphosphatidylcholine (C18:0/C10-EPC). The former was 50 times more effective as a DNA transfection agent (human umbilical artery endothelial cells) than the latter, despite their similar chemical structure and virtually identical lipoplex organization. A likely reason for the superior effectiveness of C18:1/C10-EPC relative to C18:0/C10-EPC (and to many other cationic lipoids) was suggested by the phases that evolved when these lipoids were mixed with negatively charged membrane lipid formulations. The saturated C18:0/C10-EPC remained lamellar in mixtures with biomembrane-mimicking lipid formulations [e.g., dioleoyl-phosphatidylcholine/dioleoyl-phosphatidylethanolamine/dioleoyl-phosphatidylserine/cholesterol at 45:20:20:15 (wt/wt)]; in contrast, the unsaturated C18:1/C10-EPC exhibited a lamellar-nonlamellar phase transition in such mixtures, which took place at physiological temperatures, approximately 37 degrees C. As is well known, lipid vehicles exhibit maximum leakiness and contents release in the vicinity of phase transitions, especially those involving nonlamellar phase formation. Moreover, nonlamellar phase-forming compositions are frequently highly fusogenic. Indeed, FRET experiments showed that C18:1/C10-EPC exhibits lipid mixing with negatively charged membranes that is several times more extensive than that of C18:0/C10-EPC. Thus, C18:1/C10-EPC lipoplexes are likely to easily fuse with membranes, and, as a result of lipid mixing, the resultant aggregates should exhibit extensive phase coexistence and heterogeneity, thereby facilitating DNA release and leading to superior transfection efficiency. These results highlight the phase properties of the carrier lipid/cellular lipid mixtures as a decisive factor for transfection success and suggest a strategy for the rational design of superior cationic lipid carriers.

Phase Behavior of a Phospholipid/Fatty Acid/Water Mixture Studied in Atomic Detail

Molecular dynamics simulations have been used to study the phase behavior of a dipalmitoylphosphatidylcholine (DPPC)/palmitic acid (PA)/water 1:2:20 mixture in atomic detail. Starting from a random solution of DPPC and PA in water, the system adopts either a gel phase at temperatures below approximately 330 K or an inverted hexagonal phase above approximately 330 K in good agreement with experiment. It has also been possible to observe the direct transformation from a gel to an inverted hexagonal phase at elevated temperature (approximately 390 K). During this transformation, a metastable fluid lamellar intermediate is observed. Interlamellar connections or stalks form spontaneously on a nanosecond time scale and subsequently elongate, leading to the formation of an inverted hexagonal phase. This work opens the possibility of studying in detail how the formation of nonlamellar phases is affected by lipid composition and (fusion) peptides and, thus, is an important step toward understanding related biological processes, such as membrane fusion.

CTP:Phosphocholine Cytidylyltransferase Binds Anionic Phospholipid Vesicles in a Cross-Bridging Mode

CTP:phosphocholine cytidylyltransferase (CCT) catalyzes the rate-limiting step in phosphatidylcholine (PC) synthesis, and its activity is regulated by reversible association with membranes, mediated by an amphipathic helical domain M. Here we describe a new feature of the CCTalpha isoform, vesicle tethering. We show, using dynamic light scattering and transmission electron microscopy, that dimers of CCTalpha can cross-bridge separate vesicles to promote vesicle aggregation. The vesicles contained either class I activators (anionic phospholipids) or the less potent class II activators, which favor nonlamellar phase formation. CCT increased the apparent hydrodynamic radius and polydispersity of anionic phospholipid vesicles even at low CCT concentrations corresponding to only one or two dimers per vesicle. Electron micrographs of negatively stained phosphatidylglycerol (PG) vesicles confirmed CCT-mediated vesicle aggregation. CCT conjugated to colloidal gold accumulated on the vesicle surfaces and in areas of vesicle-vesicle contact. PG vesicle aggregation required both the membrane-binding domain and the intact CCT dimer, suggesting binding of CCT to apposed membranes via the two M domains situated on opposite sides of the dimerization domain. In contrast to the effects on anionic phospholipid vesicles, CCT did not induce aggregation of PC vesicles containing the class II lipids, oleic acid, diacylglycerol, or phosphatidylethanolamine. The different behavior of the two lipid classes reflected differences in measured binding affinity, with only strongly binding phospholipid vesicles being susceptible to CCT-induced aggregation. Our findings suggest a new model for CCTalpha domain organization and membrane interaction, and a potential involvement of the enzyme in cellular events that implicate close apposition of membranes.

Molecular Mechanisms of Membrane Perturbation by Antimicrobial Peptides and the Use of Biophysical Studies in the Design of Novel Peptide Antibiotics

Antibiotic resistant bacterial strains represent a global health problem with a strong social and economic impact. Thus, there is an urgent need for the development of antibiotics with novel mechanisms of action. There is currently an extensive effort to understand the mode of action of antimicrobial peptides which are considered as one alternative to classical antibiotics. The main advantage of this class of substances, when considering bacterial resistance, is that they rapidly, within minutes, kill bacteria. Antimicrobial peptides can be found in every organism and display a wide spectrum of activity. Hence, the goal is to engineer peptides with an improved therapeutic index, i.e. high efficacy and target specificity. For the rational design of such novel antibiotics it is essential to elucidate the molecular mechanism of action. Biophysical studies have been performed using to a large extent membrane model systems demonstrating that there are distinctive different mechanisms of bacterial killing by antimicrobial peptides. One can distinguish between peptides that permeabilize and/or disrupt the bacterial cell membrane and peptides that translocate through the cell membrane and interact with a cytosolic target. Lantibiotics exhibit specific mechanisms, e.g. binding to lipid II, a precursor of the peptidoglycan layer, either resulting in membrane rupture by pore formation or preventing cell wall biosynthesis. The classical models of membrane perturbation, pore formation and carpet mechanism, are discussed and related to other mechanisms that may lead to membrane dysfunction such as formation of lipid-peptide domains or membrane disruption by formation of non-lamellar phases. Emphasis is on the role of membrane lipid composition in these processes and in the translocation of antimicrobial peptides.

Membrane restructuring by Bordetella pertussis adenylate cyclase toxin, a member of the RTX toxin family.

Adenylate cyclase toxin (ACT) is secreted by Bordetella pertussis, the bacterium causing whooping cough. ACT is a member of the RTX (repeats in toxin) family of toxins, and like other members in the family, it may bind cell membranes and cause disruption of the permeability barrier, leading to efflux of cell contents. The present paper summarizes studies performed on cell and model membranes with the aim of understanding the mechanism of toxin insertion and membrane restructuring leading to release of contents. ACT does not necessarily require a protein receptor to bind the membrane bilayer, and this may explain its broad range of host cell types. In fact, red blood cells and liposomes (large unilamellar vesicles) display similar sensitivities to ACT. A varying liposomal bilayer composition leads to significant changes in ACT-induced membrane lysis, measured as efflux of fluorescent vesicle contents. Phosphatidylethanolamine (PE), a lipid that favors formation of nonlamellar (inverted hexagonal) phases, stimulated ACT-promoted efflux. Conversely, lysophosphatidylcholine, a micelle-forming lipid that opposes the formation of inverted nonlamellar phases, inhibited ACT-induced efflux in a dose-dependent manner and neutralized the stimulatory effect of PE. These results strongly suggest that ACT-induced efflux is mediated by transient inverted nonlamellar lipid structures. Cholesterol, a lipid that favors inverted nonlamellar phase formation and also increases the static order of phospholipid hydrocarbon chains, among other effects, also enhanced ACT-induced liposomal efflux. Moreover, the use of a recently developed fluorescence assay technique allowed the detection of trans-bilayer (flip-flop) lipid motion simultaneous with efflux. Lipid flip-flop further confirms the formation of transient nonlamellar lipid structures as a result of ACT insertion in bilayers.

Properties of polyunsaturated phosphatidylcholine membranes in the presence and absence of cholesterol.

Mixtures of cholesterol with phosphatidylcholine species containing the polyunsaturated acyl chains arachidonoyl or docosahexaenoyl were studied by (13)C magic angle spinning (MAS) NMR using both cross-polarization and direct polarization, by (31)P NMR and by differential scanning calorimetry. Several unique features of these systems were observed. The separation of cholesterol in crystalline form occurred at much lower molar fractions than with other forms of phosphatidylcholine. The crystals that were formed were sensitive to the history of the sample. At cholesterol molar fractions below 0.5, they dissolved into the membrane by sequential heating and cooling scans. With higher molar fractions of cholesterol, larger amounts of anhydrous crystals were formed after the first heating. This was accompanied by the formation of non-lamellar phases. The cholesterol crystals that were formed generally were not observed by direct polarization (13)C MAS NMR, even with delay times of 100 s. This suggests that the cholesterol crystals are in a more rigid state in mixtures with these lipids. This is in contrast with the terminal methyl group of the acyl chains that is too mobile to allow cross-polarization using 1 ms contact times.

Nonlamellar Liquid Crystalline Phases and Their Particle Formation in the Egg Yolk Phosphatidylcholine/Diolein System

The phase behavior of fully hydrated egg yolk phosphatidylcholine (EPC)/diolein (DO) mixtures was investigated by small-angle X-ray scattering and 31P NMR. EPC formed a lamellar phase, whereas DO gave a negative curvature to the lipid membranes, leading to nonlamellar phase formation. At an EPC/DO molar ratio of 8:2, the mixture formed a bicontinuous cubic phase of primitive type. A bicontinuous cubic phase of diamond type was also observed at the ratio of 7:3, but it coexisted with an inverted hexagonal (HII) phase. The mixture with higher DO fractions formed the HII phase. We further investigated if these nonlamellar phases could be dispersed by high-pressure emulsification with Pluronic F127, to form cubosomes or hexosomes, as have been obtained in monoolein-based cubic and HII phases (Langmuir 2001, 17, 3917−3922; Langmuir 2002, 18, 9283−9288). As a result, cubosomes could not be obtained and vesicles were formed instead, presumably due to the existence of F127, which gave a positive curvature to the negatively curved membranes. However, the HII phase, which was less compatible with F127, could be dispersed to form hexosomes.

The kinetics of non-lamellar phase formation in DOPE-Me: relevance to biomembrane fusion.

The mechanism of the lamellar/inverted cubic (QII) phase transition is related to that of membrane fusion in lipid systems. N-Monomethylated dioleoylphosphatidylethanolamine (DOPE-Me) exhibits this transition and is commonly used to investigate the effects of exogenous substances, such as viral fusion peptides, on the mechanism of membrane fusion. We studied DOPE-Me phase behavior as a first step in evaluating the effects of membrane-spanning peptides on inverted phase formation and membrane fusion. These measurements show that: a) the onset temperatures for QII and inverted hexagonal (HII) phase formation both are temperature scan rate-dependent; b) longer pre-incubation times at low temperature and lower temperature scan rates favor formation of the QII phase; and c) in temperature-jump experiments between 61 and 65 degrees C, the meta-stable HII phase forms initially, and disappears slowly while the QII phase develops. These observations are rationalized in the context of a mechanism for both the lamellar/non-lamellar phase transition and the related process of membrane fusion.

MSI-78, an Analogue of the Magainin Antimicrobial Peptides, Disrupts Lipid Bilayer Structure via Positive Curvature Strain

In this work, we present the first characterization of the cell lysing mechanism of MSI-78, an antimicrobial peptide. MSI-78 is an amphipathic alpha-helical peptide designed by Genaera Corporation as a synthetic analog to peptides from the magainin family. (31)P-NMR of mechanically aligned samples and differential scanning calorimetry (DSC) were used to study peptide-containing lipid bilayers. DSC showed that MSI-78 increased the fluid lamellar to inverted hexagonal phase transition temperature of 1,2-dipalmitoleoyl-phosphatidylethanolamine indicating the peptide induces positive curvature strain in lipid bilayers. (31)P-NMR of lipid bilayers composed of MSI-78 and 1-palmitoyl-2-oleoyl-phosphatidylethanolamine demonstrated that the peptide inhibited the fluid lamellar to inverted hexagonal phase transition of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine, supporting the DSC results, and the peptide did not induce the formation of nonlamellar phases, even at very high peptide concentrations (15 mol %). (31)P-NMR of samples containing 1-palmitoyl-2-oleoyl-phosphatidylcholine and MSI-78 revealed that MSI-78 induces significant changes in the bilayer structure, particularly at high peptide concentrations. At lower concentrations (1-5%), the peptide altered the morphology of the bilayer in a way consistent with the formation of a toroidal pore. Higher concentrations of peptide (10-15%) led to the formation of a mixture of normal hexagonal phase and lamellar phase lipids. This work shows that MSI-78 induces significant changes in lipid bilayers via positive curvature strain and presents a model consistent with both the observed spectral changes and previously published work.

Interaction between β-Purothionin and Dimyristoylphosphatidylglycerol:A 31P-NMR and Infrared Spectroscopic Study

The interaction of β-purothionin, a small basic and antimicrobial protein from the endosperm of wheat seeds, with multilamellar vesicles of dimyristoylphosphatidylglycerol (DMPG) was investigated by 31P solid-state NMR and infrared spectroscopy. NMR was used to study the organization and dynamics of DMPG in the absence and presence of β-purothionin. The results indicate that β-purothionin does not induce the formation of nonlamellar phases in DMPG. Two-dimensional exchange spectroscopy shows that β-purothionin decreases the lateral diffusion of DMPG in the fluid phase. Infrared spectroscopy was used to investigate the perturbations, induced by β-purothionin, of the polar and nonpolar regions of the phospholipid bilayers. At low concentration of β-purothionin, the temperature of the gel-to-fluid phase transition of DMPG increases from 24°C to ∼33°C, in agreement with the formation of electrostatic interactions between the cationic protein and the anionic phospholipid. At higher protein concentration, the lipid transition is slightly shifted toward lower temperature and a second transition is observed below 20°C, suggesting an insertion of the protein in the hydrophobic core of the lipid bilayer. The results also suggest that the presence of β-purothionin significantly modifies the lipid packing at the surface of the bilayer to increase the accessibility of water molecules in the interfacial region. Finally, orientation measurements indicate that the α-helices and the β-sheet of β-purothionin have tilt angles of ∼60° and 30°, respectively, relative to the normal of the ATR crystal.

Organotin compounds promote the formation of non-lamellar phases in phosphatidylethanolamine membranes

Organotin compounds are important contaminants in the environment. They are membrane active molecules with broad biological toxicity. We have studied the interaction of tri- n-butyltin chloride and tri- n-phenyltin chloride with model membranes composed of different phosphatidylethanolamines using differential scanning calorimetry, X-ray diffraction, 31P-nuclear magnetic resonance and infrared spectroscopy. Organotin compounds laterally segregate in phosphatidylethanolamine membranes without affecting the shape and position of the lamellar gel to lamellar liquid–crystalline phase transition thermogram of the phospholipid. This is in contrast with their reported effect on phosphatidylcholine membranes [Chicano et al. (2001) Biochim. Biophys. Acta 1510, 330–341] and emphasises the importance of the nature of the lipid headgroup in determining how the behaviour of lipid molecules is affected by these toxicants. Interestingly, we have found that organotin compounds disrupt the pattern of hydrogen-bonding in the interfacial region of dielaidoylphosphatidylethanolamine membranes and have the ability to promote the formation of hexagonal H II structures in this system. These results open the possibility that some of the specific toxic effects of organotin compounds might be exerted through the alteration of membrane function produced by their interaction with the lipidic component of the membrane.

The Effect of Peptide/Lipid Hydrophobic Mismatch on the Phase Behavior of Model Membranes Mimicking the Lipid Composition in Escherichia coli Membranes

The effect of hydrophobic peptides on the lipid phase behavior of an aqueous dispersion of dioleoylphosphatidylethanolamine and dioleoylphosphatidylglycerol (7:3 molar ratio) was studied by 31P NMR spectroscopy. The peptides (WALP n peptides, where n is the total number of amino acid residues) are designed as models for transmembrane parts of integral membrane proteins and consist of a hydrophobic sequence of alternating leucines and alanines, of variable length, that is flanked on both ends by tryptophans. The pure lipid dispersion was shown to undergo a lamellar-to-isotropic phase transition at ∼60°C. Small-angle x-ray scattering showed that at a lower water content a cubic phase belonging to the space group Pn3m is formed, suggesting also that the isotropic phase in the lipid dispersion represents a cubic liquid crystalline phase. It was found that the WALP peptides very efficiently promote formation of nonlamellar phases in this lipid system. At a peptide-to-lipid (P/L) molar ratio of 1:1000, the shortest peptide used, WALP16, lowered the lamellar-to-isotropic phase transition by ∼15°C. This effect was less for longer peptides. For all of the WALP peptides used, an increase in peptide concentration led to a further lowering of the phase transition temperature. At the highest P/L ratio (1:25) studied, WALP16 induced a reversed hexagonal liquid crystalline (H II) phase, while the longer peptides still promoted the formation of an isotropic phase. Peptides with a hydrophobic length larger than the bilayer thickness were found to be unable to inhibit formation of the isotropic phase. The results are discussed in terms of mismatch between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer and its consequences for lipid-protein interactions in membranes.

Tryptophan-anchored transmembrane peptides promote formation of nonlamellar phases in phosphatidylethanolamine model membranes in a mismatch-dependent manner.

To better understand the mutual interactions between lipids and membrane-spanning peptides, we investigated the effects of tryptophan-anchored hydrophobic peptides of various lengths on the phase behavior of 1,2-dielaidoylphosphatidylethanolamine (DEPE) dispersions, using (31)P nuclear magnetic resonance and small-angle X-ray diffraction. Designed alpha-helical transmembrane peptides (WALPn peptides, with n being the total number of amino acids) with a hydrophobic sequence of leucine and alanine of varying length, bordered at both ends by two tryptophan membrane anchors, were used as model peptides and were effective at low concentrations in DEPE. Incorporation of 2 mol % of relatively short peptides (WALP14-17) lowered the inverted hexagonal phase transition temperature (T(H)) of DEPE, with an efficiency that seemed to be independent of the extent of hydrophobic mismatch. However, the tube diameter of the H(II) phase induced by the peptides was clearly dependent on mismatch and decreased with shorter peptide length. Longer peptides (WALP19-27) induced a cubic phase, both below and above T(H). Incorporation of WALP27, which is significantly longer than the DEPE bilayer thickness, did not stabilize the bilayer. The longest peptide used, WALP31, hardly affected the lipid's phase behavior, and appeared not to incorporate into the bilayer. The consequences of hydrophobic mismatch between peptides and lipids are therefore more dramatic with shorter peptides. The data allow us to suggest a detailed molecular model of the mechanism by which these transmembrane peptides can affect lipid phase behavior.