Effects of cholesterol on the interaction of Ca2(+)-ATPase with 1-palmitoyl-2-oleoylphosphatidylethanolamine. An FTIR study

Ca2(+)-ATPase from rabbit skeletal muscle has been isolated, purified, and reconstituted into vesicles containing binary mixtures of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE)/cholesterol. Fourier transform infrared spectroscopy (FTIR) was used to investigate the effect of protein on the thermotropic behavior of POPE in these reconstituted ternary complexes. The CH2 symmetric stretching modes of the phospholipid acyl chains near 2850 cm-1 served as an index of the melting process. The thermotropic transition of the POPE component in a 103:12:1 (POPE/cholesterol/Ca2(+)-ATPase) complex was shifted to lower temperatures compared with a protein-free binary lipid mixture of the same relative proportions. When combined with differential scanning calorimetric (DSC) data for the binary (POPE/cholesterol) lipid systems, this observation suggests that Ca2(+)-ATPase preferentially sequesters 15-35 molecules of POPE from the lipid mixture and therefore excludes cholesterol from its immediate environment. Higher levels of cholesterol in ternary complexes progressively eliminate the cooperative POPE melting event.     

A 2H Solid-State NMR Study of Lipid Clustering by Cationic Antimicrobial and Cell-Penetrating Peptides in Model Bacterial Membranes

Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use ²H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the β-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. ²H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct ²H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ∼40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ∼80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.     

Bilayer thickness modulates the conductance of the BK channel in model membranes

The conductance of the BK channel was evaluated in reconstituted bilayers made of POPE/POPS (3.3:1), or POPE/POPS with an added 20% of either SPM (3.3:1:1), CER (3.3:1:1), or CHL (3.3:1:1). The presence of SPM, which is known to increase bilayer thickness, significantly reduced the conductance of the BK channel. To directly test the role of membrane thickness, the conductance of the BK channel was measured in bilayers formed from PCs with acyl chains of increasing length (C14:1-C24:1), all in the absence of SPM. Slope conductance was maximal at a chain length of (C18:1) and much reduced for both thinner (C14:1) and thicker (C24:1) bilayers, indicating that membrane thickness alone can modify slope conductance. Further, in a simplified binary mixture of DOPE/SPM that forms a confined, phase-separated bilayer, the measured conductance of BK channels shows a clear bimodal distribution. In contrast, the addition of CER, which has an acyl chain structure similar to SPM but without its bulky polar head group to POPE/POPS, was without effect, as was the addition of CHL. The surface structure of membranes made from these same lipid mixtures was examined with AFM. Incorporation of both SPM and CER resulted in the formation of microdomains in POPE/POPS monolayers, but only SPM promoted a substantial increase in the amount of the high phase observed for the corresponding bilayers. The addition of CHL to POPE/POPS eliminated the phase separation observed in the POPE/POPS bilayer. The decrease in channel conductance observed with the incorporation of SPM into POPE/POPS membranes was, therefore, attributed to larger SPM-rich domains that appear thicker than the neighboring bilayer.     

A H Solid-State NMR Study of Lipid Clustering by Cationic Antimicrobial and Cell-Penetrating Peptides in Model Bacterial Membranes

Domain formation in bacteria-mimetic membranes due to cationic peptide binding was recently proposed based on calorimetric data. We now use ²H solid-state NMR to critically examine the presence and absence of domains in bacterial membranes containing zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (POPE) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) lipids. Chain-perdeuterated POPE and POPG are used in single-component membranes, binary POPE/POPG (3:1) membranes, and membranes containing one of four cationic peptides: two antimicrobial peptides (AMPs) of the β-hairpin family of protegrin-1 (PG-1), and two cell-penetrating peptides (CPPs), HIV TAT and penetratin. ²H quadrupolar couplings were measured to determine the motional amplitudes of POPE and POPG acyl chains as a function of temperature. Homogeneously mixed POPE/POPG membranes should give the same quadrupolar couplings for the two lipids, whereas the presence of membrane domains enriched in one of the two lipids should cause distinct ²H quadrupolar couplings that reflect different chain disorder. At physiological temperature (308 K), we observed no or only small coupling differences between POPE and POPG in the presence of any of the cationic peptides. However, around ambient temperature (293 K), at which gel- and liquid-crystalline phases coexist in the peptide-free POPE/POPG membrane, the peptides caused distinct quadrupolar couplings for the two lipids, indicating domain formation. The broad-spectrum antimicrobial peptide PG-1 ordered ∼40% of the POPE lipids while disordering POPG. The Gram-negative selective PG-1 mutant, IB549, caused even larger differences in the POPE and POPG disorder: ∼80% of POPE partitioned into the ordered phase, whereas all of the POPG remained in the disordered phase. In comparison, TAT rigidified POPE and POPG similarly in the binary membrane at ambient temperature, indicating that TAT does not cause dynamic heterogeneity but interacts with the membrane with a different mechanism. Penetratin maintained the POPE order but disordered POPG, suggesting moderate domain separation. These results provide insight into the extent of domain formation in bacterial membranes and the possible peptide structural requirements for this phenomenon.     

Pulmonary collectins selectively permeabilize model bacterial membranes containing rough lipopolysaccharide

We have reported that Gram-negative organisms decorated with rough lipopolysaccharide (LPS) are particularly susceptible to the direct antimicrobial actions of the pulmonary collectins, surfactant proteins A (SP-A) and D (SP-D). In this study, we examined the lipid and LPS components required for the permeabilizing effects of the collectins on model bacterial membranes. Liposomes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), with or without rough Escherichia coli LPS (J5), smooth E. coli LPS (B5), or cholesterol, were loaded with self-quenching probes and exposed to native or oxidatively modified SP-A. Fluorescence that resulted from permeabilization of liposomes and diffusion of dyes was assessed by microscopy or fluorimetry. Human SP-A and melittin increased the permeability of J5 LPS/POPE liposomes, but not B5 LPS/POPE liposomes or control (POPE only) liposomes. At a human SP-A concentration of 100 microg/mL, the permeability of the J5 LPS/POPE membranes increased 4.4-fold (p < 0.02) compared to the control with no added SP-A. Rat SP-A and SP-D also permeabilized the J5-containing liposomes. Incorporation of cholesterol into J5 LPS/POPE liposomes at a POPE:cholesterol molar ratio of 1:0.15 blocked human SP-A or melittin-induced permeability (p < 0.05) compared to cholesterol-free liposomes. Exposure of human SP-A to surfactant lipid peroxidation blocked the permeabilizing activity of the protein. We conclude that SP-A permeabilizes phospholipid membranes in an LPS-dependent and rough LPS-specific manner, that the effect is neither SP-A- nor species-specific, and that oxidative damage to SP-A abolishes its membrane destabilizing properties. Incorporation of cholesterol into the membrane enhances resistance to permeabilization by SP-A, most likely by increasing the packing density and membrane rigidity.     

Multilayer structures in lipid monolayer films containing surfactant protein C: effects of cholesterol and POPE

The influence of cholesterol and POPE on lung surfactant model systems consisting of DPPC/DPPG (80:20) and DPPC/DPPG/surfactant protein C (80:20:0.4) has been investigated. Cholesterol leads to a condensation of the monolayers, whereas the isotherms of model lung surfactant films containing POPE exhibit a slight expansion combined with an increased compressibility at medium surface pressure (10-30 mN/m). An increasing amount of liquid-expanded domains can be visualized by means of fluorescence light microscopy in lung surfactant monolayers after addition of either cholesterol or POPE. At surface pressures of 50 mN/m, protrusions are formed which differ in size and shape as a function of the content of cholesterol or POPE, but only if SP-C is present. Low amounts of cholesterol (10 mol %) lead to an increasing number of protrusions, which also grow in size. This is interpreted as a stabilizing effect of cholesterol on bilayers formed underneath the monolayer. Extreme amounts of cholesterol (30 mol %), however, cause an increased monolayer rigidity, thus preventing reversible multilayer formation. In contrast, POPE, as a nonbilayer lipid thought to stabilize the edges of protrusions, leads to more narrow protrusions. The lateral extension of the protrusions is thereby more influenced than their height.     

Exploring membrane selectivity of the antimicrobial peptide KIGAKI using solid-state NMR spectroscopy

The designed antimicrobial peptide KIGAKIKIGAKIKIGAKI possesses enhanced membrane selectivity for bacterial lipids, such as phosphatidylethanolamine and phosphatidylglycerol. The perturbation of the bilayer by the peptide was first monitored using oriented bilayer samples on glass plates. The alignment of POPE/POPG model membranes with respect to the bilayer normal was severely altered at 4 mol% KIGAKI while the alignment of POPC bilayers was retained. The interaction mechanism between the peptide and POPE/POPG bilayers was investigated by carefully comparing three bilayer MLV samples (POPE bilayers, POPG bilayers, and POPE/POPG 4/1 bilayers). KIGAKI induces the formation of an isotropic phase for POPE/POPG bilayers, but only a slight change in the (31)P NMR CSA line shape for both POPE and POPG bilayers, indicating the synergistic roles of POPE and POPG lipids in the disruption of the membrane structure by KIGAKI. (2)H NMR powder spectra show no reduction of the lipid chain order for both POPG and POPE/POPG bilayers upon peptide incorporation, supporting the evidence that the peptide acts as a surface peptide. (31)P longitudinal relaxation studies confirmed that different dynamic changes occurred upon interaction of the peptide with the three different lipid bilayers, indicating that the strong electrostatic interaction between the cationic peptide KIGAKI and anionic POPG lipids is not the only factor in determining the antimicrobial activity. Furthermore, (31)P and (2)H NMR powder spectra demonstrated a change in membrane characteristics upon mixing of POPE and POPG lipids. The interaction between different lipids, such as POPE and POPG, in the mixed bilayers may provide the molecular basis for the KIGAKI carpet mechanism in the permeation of the membrane.     

Deuterium NMR Study of the Effect of Ergosterol on POPE Membranes

We study the effect of ergosterol on the physical properties of 1-[²H₃₁]palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) multibilayers using deuterium nuclear magnetic resonance. NMR spectra were taken as a function of temperature and ergosterol concentration up to 70 mol %. The spectral first moments show that there is a dramatic difference in the ability of ergosterol to disorder the gel phase and to order the liquid-crystalline phase of POPE membranes, an unusual behavior among lipid/sterol systems studied up to now. Further investigation of the liquid-crystalline phase shows that ergosterol (erg) increases the chain order of POPE-d31, but that this effect saturates at 10 mol % ergosterol. This is in marked contrast to the effect of cholesterol (chol) on POPE membranes: the chain order of POPE increases with cholesterol to at least 45 mol %. Moreover, we found that at higher ergosterol concentrations (>40 mol %) ergosterol decreases the POPE-d31 chain order, which, to our knowledge, has not been directly observed in other lipid/sterol systems. The temperature-composition phase diagram is presented. Finally, at all ergosterol concentrations, the chain order of liquid-crystalline-phase POPE is much smaller than that of comparable POPE/chol membranes. This implies that there is no liquid-ordered phase behavior for POPE/erg membranes.     

Exploring membrane selectivity of the antimicrobial peptide KIGAKI using solid-state NMR spectroscopy

The designed antimicrobial peptide KIGAKIKIGAKIKIGAKI possesses enhanced membrane selectivity for bacterial lipids, such as phosphatidylethanolamine and phosphatidylglycerol. The perturbation of the bilayer by the peptide was first monitored using oriented bilayer samples on glass plates. The alignment of POPE/POPG model membranes with respect to the bilayer normal was severely altered at 4 mol% KIGAKI while the alignment of POPC bilayers was retained. The interaction mechanism between the peptide and POPE/POPG bilayers was investigated by carefully comparing three bilayer MLV samples (POPE bilayers, POPG bilayers, and POPE/POPG 4/1 bilayers). KIGAKI induces the formation of an isotropic phase for POPE/POPG bilayers, but only a slight change in the ³¹P NMR CSA line shape for both POPE and POPG bilayers, indicating the synergistic roles of POPE and POPG lipids in the disruption of the membrane structure by KIGAKI. ²H NMR powder spectra show no reduction of the lipid chain order for both POPG and POPE/POPG bilayers upon peptide incorporation, supporting the evidence that the peptide acts as a surface peptide. ³¹P longitudinal relaxation studies confirmed that different dynamic changes occurred upon interaction of the peptide with the three different lipid bilayers, indicating that the strong electrostatic interaction between the cationic peptide KIGAKI and anionic POPG lipids is not the only factor in determining the antimicrobial activity. Furthermore, ³¹P and ²H NMR powder spectra demonstrated a change in membrane characteristics upon mixing of POPE and POPG lipids. The interaction between different lipids, such as POPE and POPG, in the mixed bilayers may provide the molecular basis for the KIGAKI carpet mechanism in the permeation of the membrane.     

Monounsaturated PE does not phase-separate from the lipid raft molecules sphingomyelin and cholesterol: role for polyunsaturation?

We investigated interactions of the lipid raft molecules sphingomyelin (SM) and cholesterol (CHOL) in monolayers and bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycerophosphatidylethanolamine (POPE) or 1-palmitoyl-2-docosahexaenoyl-sn-glycerophosphatidylethanolamine (PDPE) at 35 degrees C. Techniques employed were pressure-area (pi-A) isotherms generated from Langmuir-Blodgett films, solid-state (2)H and (31)P NMR spectroscopies, and differential scanning calorimetry (DSC). Condensation calculated from pi-A isotherms and reduction in the enthalpy of the gel-liquid-crystalline transition in DSC scans showed CHOL has a strong affinity for POPE, comparable to that observed between SM-CHOL. Order parameters derived from (2)H NMR spectra of the perdeuterated sn-1 chain of POPE-d(31) increased by >50% upon addition of equimolar CHOL to POPE-d(31)/SM (1:1 mol) bilayers. Close proximity of CHOL to POPE even in the presence of SM is indicated. Chemical shift anisotropy (Deltasigma(csa)) measured from (1)H-decoupled (31)P NMR spectra also implied intimate lipid mixing in POPE/SM/CHOL (1:1:1 mol). In contrast, pi-A isotherms and corroborating DSC studies of PDPE/SM (1:1 mol) indicate phase separation between SM and PDPE, which was maintained in the presence of CHOL. The cholesterol-associated increase in order of the perdeuterated sn-1 chain of PDPE determined by (2)H NMR was 2-fold less for PDPE-d(31)/SM/CHOL (1:1:1 mol) than POPE-d(31)/SM/CHOL (1:1:1 mol). Our findings support the notion that acyl chain dependent lateral phase separation occurs in the presence of a docosahexaenoic acid (DHA)-containing phospholipid (PDPE), but not an oleic acid-containing phospholipid (POPE). We propose that monounsaturated lipids do not promote formation of stable lipid rafts and that polyunsaturation may be important for raft stability.     

Phosphatidylethanolamine-phosphatidylglycerol bilayer as a model of the inner bacterial membrane

Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) are the main lipid components of the inner bacterial membrane. A computer model for such a membrane was built of palmitoyloleoyl PE (POPE) and palmitoyloleoyl PG (POPG) in the proportion 3:1, and sodium ions (Na+) to neutralize the net negative charge on each POPG (POPE-POPG bilayer). The bilayer was simulated for 25 ns. A final 10-ns trajectory fragment was used for analyses. In the bilayer interfacial region, POPEs and POPGs interact readily with one another via intermolecular hydrogen (H) bonds and water bridges. POPE is the main H-bond donor in either PEPE or PEPG H-bonds; PGPG H-bonds are rarely formed. Almost all POPEs are H-bonded and/or water bridged to either POPE or POPG but PE-PG links are favored. In effect, the atom packing in the near-the-interface regions of the bilayer core is tight. Na+ does not bind readily to lipids, and interlipid links via Na+ are not numerous. Although POPG and POPE comprise one bilayer, their bilayer properties differ. The average surface area per POPG is larger and the average vertical location of the POPG phosphate group is lower than those of POPE. Also, the alkyl chains of POPG are more ordered and less densely packed than the POPE chains. The main conclusion of this study is that in the PE-PG bilayer PE interacts more strongly with PG than with PE. This is a likely molecular-level event behind a regulating mechanism developed by the bacteria to control its membrane permeability and stability consisting in changes of the relative PG/PE concentration in the membrane.     

Phase properties of liquid-crystalline Phosphatidylcholine/Phosphatidylethanolamine bilayers revealed by fluorescent probes.

The mixing properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were examined in liquid-crystalline phase using fluorescent probes incorporated into lipid bilayers. The excimer to monomer (E/M) fluorescence ratio of 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (PPC) versus PPC concentration was higher for binary mixtures containing phosphatidylcholine (PC)/phosphatidylethanolamine (PE) (1:1) compared to PC matrix. When POPC was gradually replaced with POPE, the E/M ratio also increased suggesting the enhanced lateral mobility or the lateral enrichment of PPC into domains or both. Evidences for the PE-induced domain formation were further provided by resonance energy transfer between 2-(4, 4-difluoro-5-methyl-4-boro-3a, 4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero- 3-phospho choline and PPC, which was enhanced as a function of PE concentration, and by the polarization of 1,6-diphenyl-1,3, 5-hexatriene. In addition, PE reduced free volume and polarity of lipid bilayers as measured by the emission fluorescence of 1,2-bis PPC and 6-lauroyl-2-dimethylaminonaphthalene. When POPE analogs with a methylated head group instead of normal POPE were used, the diminished effect on the domain formation was shown in the order N-methyl PE > N,N-dimethyl PE. The results suggest that the mixing properties of POPE and POPC are not random but that lipid domains of phospholipids are formed.     

Motional narrowing of the 2H NMR spectra near the chain melting transition of phospholipid/D2O mixtures

The reduction in spectral splitting, or motional narrowing, of the deuterium spectra of D₂O/phos-pholipid mixtures near the main chain melting phase transition was studied for palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and equimolar mixtures of the two at 10% hydration. For POPC the splitting was about 1700 Hz in both the fluid and gel phases, dropping to zero near the phase transition (as reported previously). For POPE the splitting remained approximately constant above the phase transition. Below the phase transition the spectrum showed a single broad line whose linewidth varied between 100 Hz and 800 Hz. This was interpreted as being due to small domains of water within a weakly hydrated crystal. POPC:POPE (1:1) samples exhibited motional narrowing behaviour similar to that for POPC except that the splitting above the phase transition was approximately twice that below the transition. The relatively broad temperature range (20 K) of the transition is explained using a simple physical model involving lipid fluctuations near the phase transition.Abbreviations NMR Nuclear Magnetic Resonance - PC phosphatidylcholine - PE phosphatidylethanolamine - POPC Palmitoyloleoylphosphatidylcholine - POPE Palmitoyloleoylphosphatidylethanolamine - H_II Inverse hexagonal phase     

Determination of the phase behaviour of phosphatidylethanolamine admixed with other lipids and the effects of calcium chloride: implications for protein kinase C regulation

The phase behaviour of 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE) was studied by differential scanning calorimetry and 31P-NMR spectroscopy. Modulation of the phase behaviour of POPE by 1-palmitoyl-2-oleoylphosphatidylserine (POPS). 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1,2-di-olein (DOG), CaCl2, MgCl2, and combinations of these substances was studied. The bilayer-forming lipids, POPS and POPC, raise the bilayer-to-hexagonal phase-transition temperature of POPE. The POPC has a greater effect than POPS, probably because the former lipid is more miscible with POPE. Addition of 10 mM CaCl2 has little effect on the phase-transitions of POPE/POPC mixtures, but it greatly decreases the effectiveness of POPS in raising the bilayer-to-hexagonal phase-transition temperature of POPE. The effectiveness of DOG in lowering the phase-transition temperature of POPE is also greatly reduced in the presence of 10 mM CaCl2. This phenomenon may play a role in the negative feedback regulation of protein kinase C.     

Unspecific membrane protein–lipid recognition: combination of AFM imaging,force spectroscopy,DSC and FRET measurements

In this work, we will describe in quantitative terms the unspecific recognition between lactose permease (LacY) of Escherichia coli, a polytopic model membrane protein, and one of the main components of the inner membrane of this bacterium. Supported lipid bilayers of 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanolamine (POPE) and 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoglycerol (POPG) (3:1, mol/mol) in the presence of Ca²⁺ display lateral phase segregation that can be distinguished by atomic force microscopy (AFM) as well as force spectroscopy. LacY shows preference for fluid (L_α) phases when it is reconstituted in POPE : POPG (3:1, mol/mol) proteoliposomes at a lipid‐to‐protein ratio of 40. When the lipid‐to‐protein ratio is decreased down to 0.5, two domains can be distinguished by AFM. While the upper domain is formed by self‐segregated units of LacY, the lower domain is constituted only by phospholipids in gel (L_β) phase. On the one hand, classical differential scanning calorimetry (DSC) measurements evidenced the segregation of a population of phospholipids and point to the existence of a boundary region at the lipid–protein interface. On the other hand, Förster Resonance Energy Transfer (FRET) measurements in solution evidenced that POPE is selectively recognized by LacY. A binary pseudophase diagram of POPE : POPG built from AFM observations enables to calculate the composition of the fluid phase where LacY is inserted. These results are consistent with a model where POPE constitutes the main component of the lipid–LacY interface segregated from the fluid bulk phase where POPG predominates. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of Physical Parameters on the Main Phase Transition of Supported Lipid Bilayers

Supported lipid bilayers composed of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) were assembled by the vesicle fusion technique on mica and studied by temperature-controlled atomic force microscopy. The role of different physical parameters on the main phase transition was elucidated. Both mixed (POPE/POPG 3:1) and pure POPE bilayers were studied. By increasing the ionic strength of the solution and the incubation temperature, a shift from a decoupled phase transition of the two leaflets, to a coupled transition, with domains in register, was obtained. The observed behavior points to a modulation of the substrate/bilayer and interleaflet coupling induced by the environment and preparation conditions of supported lipid bilayers. The results are discussed in view of the role of different interactions in the system. The influence of the substrate on the lipid bilayers, in terms of interleaflet coupling, can also help us in understanding the possible effect that submembrane elements like the cytoskeleton might have on the structure and dynamics of biomembranes.     

Influence of antimicrobial peptides on the formation of nonlamellar lipid mesophases

We have studied the influence of four antimicrobial peptides of different secondary and ternary structure - melittin (Mel), protegrin-1 (PG-1), peptidyl-glycylleucine-carboxyamide (PGLa), and gramicidin S (GS) - on the lamellar-to-nonlamellar transition of palmitoyloleoyl phosphatidylethanolamine (POPE) applying differential scanning calorimetry and small-angle X-ray diffraction. None of the peptides studied led to the formation of an inverted hexagonal phase observed for pure POPE at high temperatures. Instead either cubic or lamellar phases were stabilized to different degrees. GS was most effective in inducing a cubic phase, whereas Mel fully stabilized the lamellar phase. The behavior of POPE in the presence of PG-1 and PGLa was intermediate to GS and Mel. In addition to the known role of membrane elasticity we propose two mechanisms, which cause stabilization of the lamellar phase: electrostatic repulsion and lipid/peptide pore formation. Both mechanisms prevent transmembrane contact required to form either an inverted hexagonal phase or fusion pores, as precursors of the cubic phase.     

Membrane models of E. coli containing cyclic moieties in the aliphatic lipid chain

Nearly all molecular dynamics simulations of bacterial membranes simplify the lipid bilayer by composing it of only one or two lipids. Previous attempts of developing a model E. coli membrane have used only 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) POPG lipids. However, an important constituent of bacterial membranes are lipids containing a cyclopropane ring within the acyl chain. We have developed a complex membrane that more accurately reflects the diverse population of lipids within E. coli cytoplasmic membranes, including lipids with a cyclic moiety. Differences between the deuterium order profile of cyclic lipids and monounsaturated lipids are observed. Furthermore, the inclusion of the cyclopropane ring decreases the surface density of the bilayer and produces a more rigid membrane as compared to POPE/POPG membranes. Additionally, the diverse acyl chain length creates a thinner bilayer which matches the hydrophobic thickness of E. coli transmembrane proteins better than the POPE/POPG bilayer. We believe that the complex lipid bilayer more accurately describes a bacterial membrane and suggest the use of it in molecular dynamic simulations rather than simple POPE/POPG membranes.     

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

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

Stable cubic phases in codispersions of glucocerebroside and palmitoyloleoylphosphatidylethanolamine

The effect of glucocerebroside (GlcCer) on the structure and thermotropic phase behavior of aqueous dispersions of palmitoyloleoylphosphatidylethanolamine (POPE) has been examined using simultaneous small-angle and wide-angle X-ray diffraction methods. Binary mixtures of GlcCer:POPE in molar ratios of 2:100, 5:100, 10:100, 20:100, 30:100, and 40:100 were examined in the temperature range 20-90 degrees C. Cubic phase has been observed in binary mixtures comprised of molar ratios greater than 5:100 in the temperature range of 60-90 degrees C upon heating at a rate of 2 degrees C/min. The cubic phase is relatively stable and coexists with inverted hexagonal or lamellar phases. It persists in the codispersions throughout subsequent cooling scans to 30 degrees C. The space group of the cubic phase is determined to be Pn3m or Pn3. The lattice constant of the Pn3m cubic phase was found to be almost constant when it coexists with lamellar liquid-crystal phase. Marked temperature-dependent changes were observed when cubic phase coexists with hexagonal phase or lamellar-gel phases. This is the first report of cubic phases formed by codispersions of glycosphingolipids and phospholipids. The mechanism of cubic phase formation and the interaction between GlcCer and POPE is discussed in terms of the putative biological functions of glycolipids.