Phosphatidylethanolamine Monolayers Research Articles

GMP affected assembly behaviors of phosphatidylethanolamine monolayers elucidated by multi-resolved SFG-VS and BAM

The interaction between nucleotide molecules and lipid molecules plays important roles in cell activities, but the molecular mechanism is very elusive. In the present study, a small but noticeable interaction between the negatively charged phosphatidylethanolamine (PE) and Guanosine monophosphate (GMP) molecules was observed from the PE monolayer at the air/water interface. As shown by the sum frequency generation (SFG) spectra and Pi-A isotherm of the PE monolayer, the interaction between the PE and GMP molecules imposes very small changes to the PE molecules. However, the Brewster angle microscopy (BAM) technique revealed that the assembly conformations of PE molecules are significantly changed by the adsorption of GMP molecules. By comparing the SFG spectra of PE monolayers after the adsorption of GMP, guanosine and guanine, it is also shown that the hydrogen bonding effect plays an important role in the nucleotide-PE interactions. These results provide fundamental insight into the structure changes during the nucleotide-lipid interaction, which may shed light on the molecular mechanism of viral infection, DNA drug delivery, and cell membrane curvature control in the brain or neurons.

Biochemical and Functional Characterization by Site-Directed Mutagenesis of a Phospholipase A2 from Scorpio maurus Venom

The study of amino acid interactions in the active site of scorpion venom phospholipases A2 could help to gain insights into the structure–function relationship and the biological activities of the enzyme. In the secreted phospholipase A2 of Scorpio maurus venom glands, Glutamate 63 and Tyrosine 122 amino acids play critical roles in the catalytic mechanism through interactions with residues around the calcium-binding loop. We constructed mutants at these positions by overexpression in Escherichia coli cells. After refolding and purification of recombinant enzymes, we studied their kinetic properties using pH-stat and monolayer techniques. The mutant Glutamate 63–Aspartate (E63D) exhibited a reduced activity, while the second mutant Tyrosine 122–Arginine (Y122R) retained some activity with a 14-fold reduction in catalytic efficiency. However, both mutants remained stable in pH values ranging from 2 to 12 whereas the double mutant D63–R122 was catalytically inactive. Comparative analysis of wild-type and mutant 3-D models showed various modifications of the hydrogen-binding network linking residues Glutamate 63 and Tyrosine 122. These modifications of interactions could explain the reduction in enzymatic activity. The kinetic behavior on phosphatidylcholine and phosphatidylethanolamine monolayers of three mutants was evaluated using a baro-stat system to assess the potential association between the hydrolysis of erythrocyte membrane phospholipids and the enzyme’s capability to penetrate phospholipid monolayers at high surface pressure. Mutants’ kinetic behaviors were similar to the wild-type form with slightly modified specific activities at high surface pressure. All mutants were more active on phosphatidylethanolamine than phosphatidylcholine films at high surface pressure. This study provided new information to further elucidate structure–function relationships of scorpion venom-secreted phospholipases A2 and the design of novel potent drug molecules.

Structural Flexibility of Proteins Dramatically Alters Membrane Stability─A Novel Aspect of Lipid-Protein Interaction.

Protein isoforms are structural variants with changes in the overall flexibility predominantly at the tertiary level. For membrane associated proteins, such structural flexibility or rigidity affects membrane stability by playing modulatory roles in lipid-protein interaction. Herein, we investigate the protein chain flexibility mediated changes in the mechanistic behavior of phospholipid model membranes in the presence of two well-known isoforms, erythroid (ER) and nonerythroid (NER) spectrin. We show dramatic alterations of membrane elasticity and stability induced by spectrin in the Langmuir monolayers of phosphatidylocholine (PC) and phosphatidylethanolamine (PE) by a combination of isobaric relaxation, surface pressure-area isotherm, X-ray scattering, and microscopy measurements. The NER spectrin drives all monolayers to possess an approximately equal stability, and that required 25-fold increase and 5-fold decrease of stability in PC and PE monolayers, respectively. The untilting transition of the PC membrane in the presence of NER spectrin observed in X-ray measurements can explain better membrane packing and stability.

N-terminal acetylation of a mastoparan-like peptide enhances PE/PG segregation in model membranes

The synthetic peptides L1A and its acetylated analog (acL1A) display potent Gram-negative bactericidal activities without being hemolytic. We have gathered evidence that the N-terminal acetylation of L1A enhances the lytic activity in anionic vesicles with high capability to insert into and disturb lipid packing of model membranes. Here, the impact of L1A and acL1A was evaluated on a model membrane that mimics the cytoplasmic membrane of Gram-negative bacteria, which is rich in phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), using 3:1 mixture of POPE/DOPG and a variety of techniques. We followed peptide adsorption and penetration by zeta potential determination of large unilamellar vesicles, accessibility of tryptophan residue to acrylamide by quenching assays, and Gibbs isotherms. The secondary structure of the peptide on the membranes was assessed using circular dichroism. Peptide mixing ability with the lipids and phase segregation was assessed by the observation of Langmuir monolayers with fluorescence microscopy, as well as with differential scanning calorimetry thermograms of multilamellar vesicles. All in all, the results indicate that both peptides adsorb and penetrate POPE/DOPG membranes with similar affinities, decreasing the surface charge, and adopting alpha structures. Both peptides mix with DOPG and demix from POPE, and consequently, persist at the interface to larger surface pressures in the presence of PG than in pure PE monolayers. This selective degree of mixing of the peptides with PE and PG leads to peptide-induced segregation of PG from PE, being the less charged peptide, acL1A, able to segregate the lipids more efficiently.

-Identification of a novel intestinal phospholipase A2 from annular seabream: Insights into its catalytic mechanism and its role in biological processes

Phospholipase A2 (PLA2) is responsible for the lipid hydrolysis process. Fish PLA2 have warranted renewed interest due to their excellent properties in phospholipid digestion. We report for the first time the catalytic properties of a PLA2 secreted from the intestine of the annular seabream Diplodus annularis (IDaPLA2). The refolded IDaPLA2 was purified to homogeneity and showed a molecular mass of around 15 kDa attested by SDS-PAGE and MALDI-TOF analyses. Interestingly, IDaPLA2 revealed higher thermostability compared to mammal pancreatic sPLA2 as it was active and stable at 55 °C with specific activity of 290 U mg−1 on phosphatidylcholine (PC) as a substrate. Using the lipid monolayer technique, the activity of IDaPLA2 was found to be 21.68, 6.88 and 5.66 mol cm−2 min−1 mM−1 using phosphatidylglycerol (PG), PC and phosphatidylethanolamine (PE) monolayers, respectively, at surface pressures from 20−30 mN m−1. Interestingly, the interfacial activity of IDaPLA2 measured at higher surface pressures may highlight its ability to penetrate into phospholipid monolayers suggesting its involvement in cell lipid membrane degradation which can explain the cytotoxicity potential towards macrophage. The docking simulation data provided insights into the involvement of some key amino-acids in substrate binding and selectivity. The dynamic simulation proved the high stability of IDaPLA2. Overall, these results provide original evidence on the involvement of IDaPLA2 into the lipid hydrolysis suggesting it as a potential target in biotechnological applications.

Destabilizing effect of perfluorodecanoic acid on simple membrane models.

Perfluoroalkyl acids (PFA) are amphiphilic surfactants widely used in industry with several commercial applications. An important feature of these compounds is their non-biodegradability and their tendency to bio-accumulate in the environment, which has led to these compounds being considered among the most persistent pollutants worldwide. Many studies have provided evidence of their toxic effect on humans and wildlife. For this reason, more and more efforts have been made to better understand the effect of these compounds on living organisms. The aim of the present study is to understand how the electrostatic interactions and film compactness of biological membrane models modulate their interaction with PFA, more specifically with perfluorodecanoic acid (PFD). Langmuir isotherms and Brewster angle microscopy (BAM) are used to evaluate the effect of PFD on lipid membrane models (air/water monolayers and vesicles), analyzing the behavior of PFD : lipid mixtures. The lipids used in this study are distearoyl phosphatidic acid (DSPA), dilauroyl phosphatidic acid (DLPA) and distearoyl phosphatidylethanolamine (DSPE). PFD induces an increase in the mean molecular area per lipid in monolayers, mainly at lower surface pressures. BAM images demonstrate that PFD mixes with DLPA, inducing a decrease in gray level, while it forms a non-miscible mixture with DSPA, segregating PFD domains. Insertion studies of PFD within monolayers and dynamic light scattering experiments demonstrate that PFD can penetrate into monolayers and bilayers above 30 mN m-1, which is the lateral pressure value accepted for a cellular bilayer.

Phenylalanine intercalation parameters for liquid-disordered phase domains \u2013 a membrane model study

BackgroundPropensity of phenylalanine (Phe) for nonpolar environments drives its intercalation into phospholipid membranes, which has been implicated in metabolic and neurological disorders. The knowledge of Phe intercalation parameters can be instrumental in understanding various membrane processes triggered by interactions with Phe, in particular the early events leading to the formation of nucleation/docking sites for the self-assembly of Phe amyloid fibrils at the membrane surface.ResultsIn this study, we used monolayers of phosphatidylethanolamine (DPPE) and phosphatidylcholine (DPPC) to mimic the membrane outer leaflet. Its initial interaction with Phe was modeled by injecting Phe into the aqueous phase underneath the monolayer. Constant pressure insertion assays augmented with epifluorescence microscopy imaging were used to monitor Phe intercalation. Our primary goal was to determine the Phe intercalation area, APhe. Two values were obtained for APhe, 33 ± 2 and 48 ± 3 Å2.ConclusionsPhe appeared to discriminate between DPPE and DPPC packing, and use two modes of intercalation. The area of APhe 33 ± 2 Å2 is consistent with a Phe monomer intercalating into membrane by inserting the phenyl ring nearly normal to the membrane surface. This mode has been found to operate in DPPE membranes. For DPPC membranes however, the value of APhe = 48 ± 3 Å2 suggests that, from saline, Phe tends to intercalate as a larger species plausibly dragging along a counterion, Na+, in a Na+(Phe) complex.

Mixed Phosphoinositide/Lipid Langmuir Films in the Presence of Bivalent Cations

Local accumulation of phosphoinositides (domain formation, phosphoinositide gradients) have been linked to a broad range of physiological processes. The interaction of phosphoinositides (PIPs) with bivalent cations, cationic peptides or cationic patches in proteins has been shown to promote formation of PIP enriched domains. Our study aims to investigate how the cation/PIP interaction is modulated by the presence of lipids capable of forming hydrogen bonds with PIPs, namely phosphatidylethanolamine (PE), phosphatidylinositol (PI) or cholesterol (chol).We have investigated mixed phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)/PE monolayers at the air/water interface (Langmuir films) in the presence and absence of calcium. Both 1:1 and 4:1 mixtures of PE:PI(4,5)P2 in the absence of calcium lead to an expansion of the monolayer while the addition of relatively small amounts of calcium lead to a significant condensation. While for DOPC/PI(4,5)P2 monolayers epifluorescence microscopy revealed domain formation at low surface pressures in the presence of Ca2+, DOPE/PI(4,5)P2 monolayer did not exhibit domains even in the presence of Ca2+. These data in combination with previously published results (Graber et al. Chem. Phys. Lipids 165 (2012) 696) suggest that PE in the absence of Ca2+ inserts between PI(4,5)P2 and forms hydrogen bonds with the PI(4,5)P2 headgroup. The addition of Ca2+ to this PI(4,5)P2/PE complex results in a further condensation, presumably due to a Ca2+ bridging of the PI(4,5)P2 headgroups. These results will be compared to those obtained for PE/PI and PI/PI(4,5)P2 monolayers.

Correction: Assembly and relaxation behaviours of phosphatidylethanolamine monolayers investigated by polarization and frequency resolved SFG-VS.

Correction for 'Assembly and relaxation behaviours of phosphatidylethanolamine monolayers investigated by polarization and frequency resolved SFG-VS' by Feng Wei et al., Phys. Chem. Chem. Phys., 2015, 17, 25114-25122.

Assembly and relaxation behaviours of phosphatidylethanolamine monolayers investigated by polarization and frequency resolved SFG-VS.

The assembly conformation and kinetics of phosphatidylethanolamine (PE) lipids are the key to their membrane curvatures and activities, such as exocytosis, endocytosis and Golgi membrane fusion. In the current study, a polarization and frequency resolved (bandwidth ≈ 1 cm(-1)) picosecond sum frequency generation (SFG) system was developed to characterize phosphatidylethanolamine monolayers. In addition to obtaining π-A isotherms and Brewster angle microscopy (BAM) images, the conformational changes and assembly behaviors of phosphatidylethanolamine molecules are investigated by analyzing the SFG spectra collected at various surface pressures (SPs). The compression kinetics and relaxation kinetics of phosphatidylethanolamine monolayers are also reported. The conformational changes of PE molecules during the monolayer compression are separated into several stages: reorientation of the head group PO2(-) in the beginning of the liquid-expanded (LE) phase, conformational changes of head group alkyl chains in the LE phase, and conformational changes of tail group alkyl chains in the LE-liquid condensed (LE-LC) phase. Such an understanding may help researchers to effectively control the lipid molecular conformation and membrane curvatures during the exocytosis/endocytosis processes.

Monitoring phases and phase transitions in phosphatidylethanolamine monolayers using active interfacial microrheology.

Active interfacial microrheology is a sensitive tool to detect phase transitions and headgroup order in phospholipid monolayers. The re-orientation of a magnetic nickel nanorod is used to explore changes in the surface rheology of 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), which differ by two CH2 groups in their alkyl chains. Phosphatidylethanolamines such as DLPE and DMPE are a major component of cell membranes in bacteria and in the nervous system. At room temperature, DLPE has a liquid expanded (LE) phase for surface pressure, Π < ∼38 mN m(-1); DMPE has an LE phase for Π < ∼7 mN m(-1). In their respective LE phases, DLPE and DMPE show no measurable change in surface viscosity with Π, consistent with a surface viscosity <10(-9) N s m(-1), the resolution of our technique. However, there is a measurable, discontinuous change in the surface viscosity at the LE to liquid condensed (LC) transition for both DLPE and DMPE. This discontinuous change is correlated with a significant increase in the surface compressibility modulus (or isothermal two-dimensional bulk modulus). In the LC phase of DMPE there is an exponential increase in surface viscosity with Π consistent with a two-dimensional free area model. The second-order LC to solid (S) transition in DMPE is marked by an abrupt onset of surface elasticity; there is no measurable elasticity in the LC phase. A measurable surface elasticity in the S phase suggests a change in the molecular ordering or interactions of the DMPE headgroups that is not reflected in isotherms or in grazing incidence X-ray diffraction. This onset of measurable elasticity is also seen in DLPE, even though no indication of a LC-S transition is visible in the isotherms.

The Influence of pH on Phosphatidylethanolamine Monolayer at the Air/Aqueous Solution Interface

The dependence of the interfacial tension of a phosphatidylethanolamine (PE) monolayer on the pH of the aqueous solution has been studied. A theoretical equation is derived to describe this dependence. A simple model of the influence of pH on the phosphatidylethanolamine monolayer at the air/hydrophobic chains of PE is presented. The contributions of additive phosphatidylethanolamine forms (both interfacial tension values and molecular area values) depend on pH. The interfacial tension values and the molecular area values for PEH+ and PEOH− forms of phosphatidylethanolamine were calculated. The assumed model was verified experimentally. The experimental results agreed with those derived from the theoretical equation in a whole range of pH values.

Interactions between phosphatidylethanolamine monolayers and phenols in relation to antibacterial activity

Interactions between phosphatidylethanolamine monolayers and phenols in relation to antibacterial activity

Proceedings: Interactions of 4-chlorophenol and phenol with phosphatidylethanolamine monolayers in relation to antibacterial action.

Proceedings: Interactions of 4-chlorophenol and phenol with phosphatidylethanolamine monolayers in relation to antibacterial action.

Monomolecular layers of synthetic phosphatides.

Abstract In studying the functions of phosphatides in biological membranes, investigations have been made into monomolecular layers of synthetic phosphatides. Force-area curves of monolayers of phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, all containing identical fatty acids, showed small differences, obviously to be attributed to differences in size and charge of the end groups. The shifts of the force-area curves within one class of phosphatides were more pronounced and are brought about by variations of the apolar moiety. Shortening of the chain length and particularly unsaturation of the fatty acid constituents greatly expanded the films of the l-α-lecithins, thereby increasing the closest stable packing attainable. Force-area curves of structurally isomeric mixed-acid l-α-phosphatides, carrying dissimilar fatty acids in different positions, were identical. Mixed films consisting of phosphatides and cholesterol in molar equivalents—at proportions also found in red cell membranes—revealed a condensing effect of cholesterol on the film of phosphatides containing certain unsaturated fatty acid constituents.

Duramycin-Induced Destabilization of a Phosphatidylethanolamine Monolayer at the Air−Water Interface Observed by Vibrational Sum-Frequency Generation Spectroscopy

Duramycin is a small tetracyclic peptide which binds specifically to ethanolamine phospholipids (PE). In this study, we used lipid monolayers consisting of 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) and various phosphatidylcholines (PC) to investigate the effect of duramycin on the organization of lipids and its influence on surrounding water molecules, using vibrational sum-frequency generation spectroscopy in conjunction with surface pressure measurements and fluorescence microscopy. The results show that while duramycin has no effect on the PC lipid monolayers, it induces significant disorder of PE molecules and causes an increase of the PE monolayer surface pressure. Duramycin adopts a β-sheet conformation and is well-ordered at the air-water interface as well as after binding to PE. Our results are consistent with duramycin inserting into the PE monolayer via its hydrophobic end, exposing phenylalanine residues to the lipid. Binding of duramycin to PE broadens the hydrogen-bond distribution of lipid-bound water molecules, notably increasing the fraction of the less strongly hydrogen-bonded, possibly undercoordinated, water molecules. Fluorescence microscopy reveals that the interaction of duramycin with PE causes a change in the shape of the liquid-condensed domains of the PE monolayer from circular to horseshoe-like, indicating a reduction of line tension at the boundary of the two lipid phases. These results reveal that the first steps in the disruption of membrane integrity by duramycin consist of a reduction of the line tension, a decrease in the lipid order, and a weakening of the hydrogen bonding network of water around PE.

ON-OFF switching of transcriptional activity of large DNA through a conformational transition in cooperation with phospholipid membrane.

We report that structural transitions of DNA cause the ON−OFF switching of transcriptional activity in cooperation with phospholipid membrane in a reconstituted artificial cell. It has been shown that long DNA of more than 20−30 kilo base-pairs exhibits a discrete conformational transition between a coiled state and highly folded states in aqueous solution, depending on the presence of various condensing agents such as polyamine. Recently, we reported a conformational transition of long DNA through interplay with phospholipid membrane, from a folded state in aqueous phase to an extended coil state on a membrane surface, in a cell-sized water-in-oil microdroplet covered by phosphatidylethanolamine monolayer (Kato, A.; Shindo, E.; Sakaue, T.; Tsuji, A.; Yoshikawa, K. Biophys. J.2009, 132, 1678−1686). In this study, to elucidate the effects of these conformational changes on the biologically important function of DNA, transcription, we investigated the transcriptional activity of DNA in a microdroplet. Transcriptional activity was evaluated at individual DNA molecule level by a method we developed, in which mRNA molecules are labeled with fluorescent oligonucleotide probes. Transcription proceeded on almost all of the DNA molecules with a coiled conformation in the aqueous phase. In the presence of a tetravalent amine, spermine, the DNA had a folded conformation, and transcription was completely inhibited. When the Mg2+ concentration was increased, DNA was adsorbed onto the inner surface of the membrane and exhibited an extended conformation. The transcription experiments showed that this conformational transition recovered transcriptional activity; transcription occurred on DNA molecules that were on the membrane.

The effect of the injection of alkanols and alkanediols beneath dipalmitoyl phosphatidylethanol amine monolayers

The energies of activation of the interaction of alkanols and alkanediols with dipalmitoyl phosphatidylethanol amine monolayers spread at the air/water interface were estimated from the increase in the surface pressure with the concentration of the injected alcohol. The energies of activation are linear functions of the chain length of the injected alkanol for the one-, two-, three-, six-, and eight-carbon chains and of the alkanediols for the two-, three- and six-carbon chains. The plots of the data that exist in the literature of the reflection coefficients of these alkanols and alkanediols against the chain length obtained in biological membranes present similar linearities.

The Equilibria of Phosphatidylethanolamine-Cholesterol and Phosphatidylcholine–Phosphatidylethanolamine in Monolayers at the Air/Water Interface

Monolayers of phosphatidylethanolamine (PE), cholesterol (Ch), phosphatidylcholine (PC), and binary mixtures (PC-PE or PE-Ch) were investigated at the air/water interface. The surface tension values of pure and mixed monolayers were used to calculate π -A isotherms. The surface tension measurements were carried out at 20°C using an improved Teflon trough and a Nima 9000 tensiometer. The Teflon trough was filled with a subphase of triple-distilled water. Known amounts of lipid dissolved in 1-chloropropane were placed at the surface using a syringe. The interactions between phosphatidylethanolamine and cholesterol as well as phosphatidylcholine and phosphatidylethanolamine result in significant deviations from the additivity rule. An equilibrium theory to describe the behavior of monolayer components at the air/water interface was developed in order to obtain the stability constants of PC-PE and PE-Ch complexes. We considered the equilibrium between the individual components and the complex and established that phosphatidylethanolamine and cholesterol as well as phosphatidylcholine and phosphatidylethanolamine formed highly stable 1:1 complexes.

Influence of surfactant protein C on the interfacial behavior of phosphatidylethanolamine monolayers

In the current work we study with monolayer tensiometry and Brewster angle microscopy (BAM) the surface properties of Dipalmitoleoylphosphatidylethanolamine (DPoPE) films at the air/water interface in presence and absence of specific surfactant protein C (SP-C). DPoPE is used, as it readily forms both lamellar (L(alpha)) and non-lamellar inverted hexagonal (H(II)) phases and appears as a suitable model phospholipid for probing the interfacial properties of distinct lipid phases. At pure air/water interface L(alpha) shows faster adsorption and better surface disintegration than H(II) phase. The interaction of DPoPE molecules with SP-C (predeposited at the interface) results in equalizing of the interfacial disintegration of the both phases (reaching approximately the same equilibrium surface tension) although the adsorption kinetics of the lamellar phase remains much faster. Monolayer compression/decompression cycling revealed that the effect of SP-C on dynamic surface tensions (gamma (max) and gamma (min)) of mixed films is remarkably different for the two phases. If gamma (max) for L(alpha) decreased from the first to the third cycle, the opposite effect is registered for H(II) where gamma (max) increases during cycling. Also the significant decrease of gamma (min) for L(alpha) in SP-C presence is not observed for H(II) phase. BAM studies reveal the formation of more uniform and homogeneously packed DPoPE monolayers in the presence of SP-C.