PE Bilayers Research Articles

Extracellular membrane vesicles derived from Komagataeibacter oboediens exposed on the International Space Station fuse with artificial eukaryotic membranes in contrast to vesicles of reference bacterium

Membranous Extracellular Vesicles (EVs) of Gram-negative bacteria are a secretion and delivery system that can disseminate bacterial products and interact with hosts and the environment. EVs of nonpathogenic bacteria deliver their contents by endocytosis into eukaryotic cells, however, no evidence exists for a fusion delivery mechanism. Here, we describe the fusion of exposed to space/Mars-like stressors simulated on the International Space Station vesicles (E-EVs) from Komagataeibacter oboediens to different types of model planar membranes in comparison with the EVs of the ground-based reference strain. The most reliable fusion was achieved with PC:PE:ergosterol or sterol-free PC:PE bilayers. The relative permeability ratio (PK+/PCl-) estimated from the shift of zero current potential according to Goldman–Hodgkin–Katz equation consisted of 4.17 ± 0.48, which coincides with preferential cation selectivity of the EV endogenous channels. The increase in membrane potential from 50 mV to 100 mV induced the fusion of E-EVs with all tested lipid compositions. The fusion of model exosomes with planar bilayer lipid membranes was confirmed by separate step-like increases in its conductance. In contrast, the ground-based reference K. oboediens EVs never induced the fusion event. In our study, we show membrane lipidome perturbations and increased protein aggregation occurred in the exposed samples in the harsh environment when outer membranes of K. oboediens acquired the capability of both homo- and heterotypic fusion possibly by altered membrane fluidity and the pore-forming capability.

Molecular structures of fluid phosphatidylethanolamine bilayers obtained from simulation-to-experiment comparisons and experimental scattering density profiles.

Following our previous efforts in determining the structures of commonly used PC, PG, and PS bilayers, we continue our studies of fully hydrated, fluid phase PE bilayers. The newly designed parsing scheme for PE bilayers was based on extensive MD simulations, and is utilized in the SDP analysis of both X-ray and neutron (contrast varied) scattering measurements. Obtained experimental scattering form factors are directly compared to our simulation results, and can serve as a benchmark for future developed force fields. Among the evaluated structural parameters, namely, area per lipid A, overall bilayer thickness DB, and hydrocarbon region thickness 2DC, the PE bilayer response to changing temperature is similar to previously studied bilayers with different headgroups. On the other hand, the reduced hydration of PE headgroups, as well as the strong hydrogen bonding between PE headgroups, dramatically affects lateral packing within the bilayer. Despite sharing the same glycerol backbone, a markedly smaller area per lipid distinguishes PE from other bilayers (i.e., PC, PG, and PS) studied to date. Overall, our data are consistent with the notion that lipid headgroups govern bilayer packing, while hydrocarbon chains dominate the bilayer's response to temperature changes.

Decoding The Molecular Basis Of Anti-Apoptotic Cardiac Ca2+ Signalling Via Human RyR2 Splice Variants

Apoptotic cell death is involved in normal and pathologic cardiac function. Apoptosis is pivotal in normal developmental cardiogenesis and yet unregulated cardiomyocyte loss via apoptosis contributes to cardiac degeneration in heart failure. There is emerging evidence that distinct populations of cardiomyocytes may exhibit differential susceptibility to apoptosis. We showed that alternative splicing of the cardiac ryanodine receptor (RyR2) modulated the susceptibility of cardiomyocytes to Ca2+-linked apoptosis (George et al., (2007)). Specifically, a 24bp splice variant (encoding VTGSQRSK inserted distal to residue 3715 in human RyR2) suppressed homeostatic and agonist-evoked Ca2+ fluxes in cardiomyocytes. We proposed that alternative splicing of RyR2 may represent a mechanism for ‘tuning’ pro- and anti-apoptotic Ca2+ fluxes. Our data supports the concept that the 24bp splice insertion stabilises RyR2-mediated Ca2+ release, and we tested this hypothesis using single channel and cellular approaches. In single channel experiments using CHAPS-solubilised, sucrose gradient-enriched preparations incorporated into PE bilayers, we investigated the effects of synthetic peptides containing VTGSQRSK on the Ca2+ sensitivity of recombinant human RyR2 channels devoid of alternatively spliced insertions (RyR2−/−). In a complementary approach we used confocal imaging to determine the effects of these peptides on Ca2+ handling in living cardiomyocytes. Peptides were injected into the nucleus and cytoplasm of synchronously-coupled HL-1 cells (that exclusively express RyR2−/−) and cellular Ca2+ fluxes were analysed using the multi-parametric Synchronicity-Amplitude-Length and Variability of Oscillation (SALVO) program that describes the ‘contractile’ and ‘non-contractile’ aspects of cellular Ca2+ handling. In both approaches, peptides encoding a splice insertion that did not protect cells from Ca2+-linked apoptosis (FAIDSLCGFG), or scrambled sequences were used as controls. Our data shows the utility of these complementary approaches in determining the mechanistic basis of altered cellular Ca2+ handling mediated by alternative splicing of RyR2.

New Evidence for TiO2 Photocatalysis during Bilayer Lipid Peroxidation

The photocatalytic peroxidation of the bovine brain l-α-phosphatidyl-ethanolamine (PE) vesicle was measured and used as a model for phospholipid peroxidation. Synthetic phosphatidyl-ethanolamine, E. coli phosphatidyl-ethanolamine, lipid polysaccharide (LPS), and cardiopilin were used to compare the TiO2 photocatalysis leading to cell wall membrane degradation. During the photocatalytic degradation, the appearance and growth kinetics were followed for (a) conjugated double bond formation, (b) malondialdehyde (MDA), (c) peroxides, and finally (d) the observed CO2 evolution. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was measured of PE bilayers cast on TiO2 films and to a lesser extent for other phospholipids during TiO2 photocatalysis. The decay kinetics induced by photocatalysis of the isolated cis CC−H (3008 cm-1), −CH2, and −CH3 groups and of the acyl-ester bond of the fatty acid in the glycerol backbone were followed in detail. The photocatalysis was also observed to induce spectral shifts in the CH2 vibrations and changes in the asymmetric phosphate ester (C−(PO4)-−C) stretching vibrations. The ATR-FTIR spectral changes suggest structural changes of the lipid bilayer due to peroxidation. The significant part of the photocatalytic peroxidation seems to take place at the TiO2 surface by heterogeneous mediated processes. But concomitantly, a homogeneous chain radical peroxidation of PE occurs in aqueous solution by a radical chain mechanism.

Hydration, Structure, and Molecular Interactions in the Headgroup Region of Dioleoylphosphatidylcholine Bilayers: An Electron Spin Resonance Study

The relationship between bilayer hydration and the dynamic structure of headgroups and interbilayer water in multilamellar vesicles is investigated by electron spin resonance methods. Temperature variations of the order parameter of a headgroup spin label DPP-Tempo in DOPC in excess water and partially dehydrated (10 wt % water) show a cusp-like pattern around the main phase transition, T c. This pattern is similar to those of temperature variations of the quadrupolar splitting of interbilayer D 2O in PC and PE bilayers previously measured by 2H NMR, indicating that the ordering of the headgroup and the interbilayer water are correlated. The cusp-like pattern of these and other physical properties around T c are suggestive of quasicritical fluctuations. Also, an increase (a decrease) in ordering of DPP-Tempo is correlated with water moving out of (into) interbilayer region into (from) the bulk water phase near the freezing point, T f. Addition of cholesterol lowers T f, which remains the point of increasing headgroup ordering. Using the small water-soluble spin probe 4-PT, it is shown that the ordering of interbilayer water increases with bilayer dehydration. It is suggested that increased ordering in the interbilayer region, implying a lowering of entropy, will itself lead to further dehydration of the interbilayer region until its lowered pressure resists further flow, i.e., an osmotic phenomenon.

Peptide models of the helical hydrophobic transmembrane segments of membrane proteins: interactions of acetyl-K2-(LA)12-K2-amide with phosphatidylethanolamine bilayer membranes.

High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of a synthetic alpha-helical hydrophobic transmembrane peptide, acetyl-Lys(2)-(Leu-Ala)(12)-Lys(2)-amide [(LA)(12)], and members of a homologous series of n-saturated diacylphosphatidylethanolamines (PEs). In the lower range of peptide mole fractions, the DSC endotherms exhibited by the lipid/peptide mixtures consist of two components. The temperature and cooperativity of the sharper, higher temperature component are very similar to those of pure PE bilayers and are almost unaffected by variations in the protein/lipid ratio. However, the fractional contribution of this component to the total enthalpy changes decreases with increases in peptide concentration, and this component completely disappears at higher protein mole fractions. The other component, which is less cooperative and occurs at a lower temperature, predominates at higher protein concentrations. These two components of the DSC endotherm have been assigned to the chain-melting phase transitions of peptide-nonassociated and peptide-associated PE molecules, respectively. Although the temperature at which the peptide-associated PE molecules melt is progressively decreased by increases in (LA)(12) concentration, the magnitude of this downward shift is progressively greater as the length of the PE hydrocarbon chain decreases. As well, mixtures of (LA)(12) with the longer chain PEs exhibit unusual biomodal enthalpy variations, suggesting peptide immiscibility in thicker gel state bilayers. Moreover, the enthalpy of the chain-melting transition of the peptide-associated PE does not decrease to zero even at high peptide concentrations, indicating that (LA)(12) attenuates but does not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. Our FTIR spectroscopic data indicate that (LA)(12) remains in a predominantly alpha-helical conformation in liquid-crystalline PE bilayers of various hydrophobic thickness but that the helical conformation is altered in gel-state PE bilayers generally, probably due to peptide lateral aggregation. These data also suggest that (LA)(12) significantly disorders the hydrocarbon chains of adjacent PE molecules in both the gel and liquid-crystalline states, relatively independently of lipid hydrocarbon chain length. Many aspects of PE/(LA)(12) interactions exhibit a different dependence on the hydrophobic thickness of the host bilayer than was observed in our previous study of (LA)(12)-phosphatidylcholine (PC) model membranes [Zhang et al. (1995) Biochemistry 34, 2362-2371]. The differing effects of (LA)(12) incorporation on PE and PC bilayers is ascribed primarily to the much stronger lipid polar headgroup interactions characteristic of the former system. Finally, the considerable differences observed in the behavior of (LA)(12) and the related polyleucine-based peptide P(24) in both PC and PE bilayers indicate that the structure of the hydrophobic core of alpha-helical transmembrane peptides can affect their conformational plasticity and state of aggregation and thus the nature of their interactions with different phospholipid bilayers.

Interaction of a peptide model of a hydrophobic transmembrane alpha-helical segment of a membrane protein with phosphatidylethanolamine bilayers: differential scanning calorimetric and Fourier transform infrared spectroscopic studies

High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of a synthetic alpha-helical hydrophobic transmembrane peptide, Acetyl-Lys2-Gly-Leu24-Lys2-Ala-Amide, and members of a homologous series of n-saturated diacylphosphatidylethanolamines (PEs). In the lower range of peptide mol fractions, the DSC endotherms exhibited by the lipid/peptide mixtures consist of two components. The temperature and cooperativity of the sharper, higher-temperature component are very similar to those of pure PE bilayers and are almost unaffected by variations in the peptide/lipid ratio. However, the fractional contribution of this component to the total enthalpy change decreases with increases in peptide concentration, and this component completely disappears at higher peptide mol fractions. The other component, which is less cooperative and occurs at a lower temperature, predominates at higher peptide concentrations. These two components of the DSC endotherm can be attributed to the chain-melting phase transitions of peptide-nonassociated and peptide-associated PE molecules, respectively. Although the temperature at which the peptide-associated PE molecules melt is progressively decreased by increases in peptide concentration, the magnitude of this shift is independent of the length of the PE hydrocarbon chain. In addition, the width of the phase transition observed at higher peptide concentrations is also relatively insensitive to PE hydrocarbon chain length, except that peptide gel-phase immiscibility occurs in very short- or very long-chain PE bilayers. Moreover, the enthalpy of the chain-melting transition of the peptide-associated PE does not decrease to 0 even at high peptide concentrations, suggesting that this peptide does not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. The FTIR spectroscopic data indicate that the peptide remains in a predominantly alpha-helical conformation, but that the peptide alpha-helix is subject to small distortions coincident with the changes in hydrophobic thickness that accompany the chain-melting phase transition of the PE bilayer. These data also indicate that the peptide significantly disorders the hydrocarbon chains of adjacent PE molecules in both the gel and liquid-crystalline states relatively independently of lipid hydrocarbon chain length. The relative independence of many aspects of PE-peptide interactions on the hydrophobic thickness of the host bilayer observed in the present study is in marked contrast to the results of our previous study of peptide-phosphatidylcholine (PC) model membranes (Zhang et al. (1992) Biochemistry 31:11579-11588), where strong hydrocarbon chain length-dependent effects were observed. The differing effects of peptide incorporation on PE and PC bilayers is ascribed to the much stronger lipid polar headgroup interactions in the former system. We postulate that the primary effect of transmembrane peptide incorporation into PE bilayers is the disruption of the relatively strong electrostatic and hydrogen-bonding interactions at the bilayer surface, and that this effect is sufficiently large to mask the effect of hydrophobic mismatch between the lengths of the hydrophobic core of the peptide and its host bilayer.

The effect of plant hormone abscisic acid on model membranes - differential scanning calorimetry and planar bilayer membranes investigations

The effect of abscisic acid on the thermotropic properties of dipalmitoylphosphatidylcholine (DPPC) and on phosphatidylethanolamines (natural (PE) and dipalmitoylphosphatidylethanolamine (DPPE)) bilayers was investigated by differential scanning calorimetry (DSC). Abscisic acid eliminates the pretransition of DPPC, causes a downward shift of its temperature of melting ( T m) and broadens the melting peak without changing the enthalpy of melting. In natural PE bilayers interacting with abscisic acid a small decrease in the enthalpy of melting almost without change of T m was detected, whereas in synthetic DPPE abscisic acid caused a small shift of T m and small broadening of the melting peak without changing the enthalpy of melting. Abscisic acid increases the conductance to Na + or K + by three orders of magnitude in planar lipid membranes formed from PE monolayers and by less than two orders of magnitude in membranes formed from PC monolayers.

Area per molecule and distribution of water in fully hydrated dilauroylphosphatidylethanolamine bilayers.

The area per lipid molecule for fully hydrated dilauroylphosphatidylethanolamine (DLPE) has been obtained in both the gel and liquid-crystalline states by combining wide-angle X-ray diffraction, electron density profiles, and previously published dilatometry results [Wilkinson, D. A., & Nagle, J. F. (1981) Biochemistry 20, 187-192]. The molecular area increases from 41.0 +/- 0.2 to 49.1 +/- 1.2 A2 upon melting from the gel to liquid-crystalline phase. The thickness of the bilayer, as measured from the electron density profiles, decreases about 4 A upon melting, from 45.2 +/- 0.3 to 41.0 +/- 0.6 A. A somewhat unexpected result is that the fluid layer between fully hydrated bilayers is the same in both gel and liquid-crystalline phases and is only about 5 A thick. From these data, plus the volume of the anhydrous DLPE molecule, it is possible to determine the number of water molecules per lipid and their approximate distribution relative to the lipid molecule. Our analysis shows that there are about 7 and 9 waters per DLPE molecule in the gel and liquid-crystalline phases, respectively. About half of the water is located in the fluid space between adjacent bilayers, and the remaining waters are intercalated into the bilayer, presumably in the head group region. There are significantly fewer water molecules in the fluid spaces between DLPE bilayers than in the fluid spaces in gel- or liquid-crystalline-phase phosphatidylcholine bilayers. This small fluid space in PE bilayers could arise from interbilayer hydrogen bond formation through the water molecules or electrostatic interactions between the amine and phosphate groups on apposing bilayers.

Influence of trinitrophenylation on the structure and dynamics of phosphatidylethanolamine-containing model membranes

The influence of trinitrophenylation on hydration, acyl chain melting characteristics, and polymorphism of phosphatidylethanolamine- (PE-) containing model membranes has been investigated. 2H nuclear magnetic resonance spectroscopy (NMR) was used to study the hydration properties of (trinitro- pheny1)phosphatidylethanolamine (TNPPE). 31P NMR techniques, including saturation transfer and the use of phosphatidylcholine thio analogues, were employed to investigate the TNPPE head-group conformation. The thermotropic behavior of PE/TNPPE mixtures was studied by means of differential scanning calorimetry. The macroscopic organization of the phospholipids was monitored by 31P NMR, small-angle X-ray diffraction, and freeze-fracture electron microscopy. The results indicate that TNPPE is anhydrous in character and does not form ordered structures by itself. Furthermore, even in excess of water, the phosphate region of the fluid TNPPE molecules cannot undergo long-axis rotation, presumably due to intermolecular ring interactions. On the other hand, in aqueous dispersions TNPPE molecules are to a limited extent ( N 20 mol 7%) soluble in PE. From the decrease in enthalpy of the gel to liquid-crystalline phase transition the TNPPE molecules appear to interact with PE molecules in a 1 :4 stoichiometry. In hydrated mixtures with phosphatidylcholine (PC) or PE, the phosphate moiety of the TNPPE molecule undergoes long-axis rotation. However, by comparison with PC and PE in these samples, TNPPE has a different head-group conformation. This is possibly caused by a trinitrophenyl ring orientation either at the bilayer/water interface or per- pendicular to the plane of the bilayer. TNPPE hardly influences the temperature of the gel to liquid-crystalline phase transition in PE bilayers. In contrast, TNPPE strongly destabilizes the bilayer organization in both PE and PC model membranes, most likely due to dehydration effects and interbilayer trinitrophenyl ring interactions.

Kinetics and thermodynamics of calcium-induced lateral phase separations in phosphatidic acid containing bilayers.

The effects of calcium on the mixing of synthetic diacylphosphatidylcholines (PC's) and diacylphosphatidylethanolamines (PE's) with the corresponding phosphatidic acids (PA's) have been examined by high-sensitivity differential scanning calorimetry and by measurements of the fluorescence of labeled PA or PC species in PA-PC bilayers. Calorimetrically derived phase diagrams for dimyristoyl- and dielaidoyl-substituted PA-PC and PA-PE mixtures indicate that these species are readily miscible in the absence of calcium but phase-separate very extensively in the presence of high levels of calcium (30 mM). The limiting solubilities of PA (Ca2+) in liquid-crystalline PC or PE bilayers are less than or equal to 10 and approximately 5 mol %, respectively, while approximately 20 mol % of PC or PE can be introduced into the "cochleate" phase of PA (Ca2+) before a distinct PC-rich (or PE-rich) phase appears. The kinetics of calcium-induced lateral phase separations were examined for dioleoyl- and dielaidoyl-substituted PA-PC unilamellar vesicles labeled with fluorescent (C12-NBD-acyl) PA or PC, whose fluorescence becomes partially quenched upon phase separation. Our results indicate that, for the PA-PC system, lateral phase separation is very rapid (approximately less than 1 s) after calcium addition and develops partially (possibly in only one face of the bilayer) when calcium is present only on one side of the bilayer. Moreover, phase separations can develop at a rate faster than that of vesicle diffusion when calcium is added to dilute suspensions of vesicles, suggesting that interbilayer contacts are not essential to promote phase separations.

Quantum-chemical and empirical calculations on phospholipids. VI: The stereospecific interaction of Na + with phosphatidylethanolamine and with phosphatidylcholine layer systems

Using 1-6-12 atom-atom potential functions with a solvent-averaged electrostatic potential U = q I q j/( ϵD( r) r 1j between the charges q I and q j the stereospecific interaction of Na + with clusters of l-glycero-phosphorylethanolamine (GPE) and -choline (GPC) in a two-dimensional planar lattice was calculated. With neutral PE headgroups only a weak attractive interaction of −140 kJ/mol was found. This should be compensated in PE/water systems by reorientational processes within the Na + hydration shell to get an optimum binding of Na + with the PE headgroup. With a phosphorylethanolamine anion within a neutral PE headgroup environment a stereospecific binding energy of −500 kJ/mol was calculated. The interaction of Na + or Ca 2+ with clusters of phosphatidylcholine is always repulsive. These results are in a very good agreement with experimental ones where a binding of Na + with PE bilayers at pH > 7.5, but not with PC bilayers was detected.