Pure DMPC Bilayer Research Articles

Experimental and theoretical studies of emodin interacting with a lipid bilayer of DMPC.

Emodin is one of the most abundant anthraquinone derivatives found in nature. It is the active principle of some traditional herbal medicines with known biological activities. In this work, we combined experimental and theoretical studies to reveal information about location, orientation, interaction and perturbing effects of Emodin on lipid bilayers, where we have taken into account the neutral form of the Emodin (EMH) and its anionic/deprotonated form (EM-). Using both UV/Visible spectrophotometric techniques and molecular dynamics (MD) simulations, we showed that both EMH and EM- are located in a lipid membrane. Additionally, using MD simulations, we revealed that both forms of Emodin are very close to glycerol groups of the lipid molecules, with the EMH inserted more deeply into the bilayer and more disoriented relative to the normal of the membrane when compared with the EM-, which is more exposed to interfacial water. Analysis of several structural properties of acyl chains of the lipids in a hydrated pure DMPC bilayer and in the presence of Emodin revealed that both EMH and EM- affect the lipid bilayer, resulting in a remarkable disorder of the bilayer in the vicinity of the Emodin. However, the disorder caused by EMH is weaker than that caused by EM-. Our results suggest that these disorders caused by Emodin might lead to distinct effects on lipid bilayers including its disruption which are reported in the literature.

The Interplay of Structure and Dynamics in Mixed Lipid Bilayers

The implication of lipids in membrane mediated processes has motivated significant theoretical and experimental research interest to understand the functional significance of lipid chemical structure variations on the biomembrane structure and dynamics. In particular, hydrophobic mismatch between lipids with different acyl chain lengths is expected to play an important role in determining the local membrane structure and collective membrane dynamics. Herein we investigated these phenomena in large unilamellar vesicles composed of binary mixtures of lipids with a 4 carbon mismatch in tail length, dimyristoylphosphatidylcholine (DMPC, 14:0 C) and distearoylphosphatidycholine (DSPC, 18:0 C), using neutron scattering techniques. Structural studies using small angle neutron scattering (SANS) revealed that the mixed lipid bilayers were thinner than expected for a simple composition-weighted average of the pure component bilayers. The structural differences were accompanied by dramatic differences in the mixed lipid membrane dynamics. Neutron spin echo spectroscopy (NSE) experiments showed that the mixed lipid bilayers were more dynamic than their single component analogs, with the mixed bilayers having a lower bending modulus and increased thickness fluctuation amplitude than the pure DMPC or DSPC bilayers. Both of these enhanced dynamics were consistent with a decrease in the area expansion modulus of the mixed systems. Together our results demonstrate the influence of lipid composition on the bilayer biophysical properties and highlight the complex interplay between structure and dynamics in lipid membranes.

Membrane Sterols Modulate the Binding Mode of Amphotericin B without Affecting Its Affinity for a Lipid Bilayer.

Membrane-active antibiotics are known to selectively target certain pathogens based on cell membrane properties, such as fluidity, lipid ordering, and phase behavior. These are in turn modulated by the composition of a lipid bilayer and in particular by the presence and type of membrane sterols. Amphotericin B (AmB), the golden standard of antifungal treatment, exhibits higher activity toward ergosterol-rich fungal membranes, which permits its use against systemic mycoses; however, the selectivity for fungal membranes is far from satisfactory leading to severe side effects. Despite decades of research, no consensus has emerged on the origin of AmB specificity for fungal cells and its actual mode of action at the molecular level. Previously, it has been proposed that the specific action of AmB is related to differences in its affinity for membranes of different composition. In this work, we investigate this relationship by employing molecular dynamics simulations to compare the free energy of insertion of AmB into three types of membranes: a pure DMPC bilayer and DMPC bilayers containing 30% of cholesterol or ergosterol. We analyze the orientation of AmB molecules within the bilayer in order to unambiguously establish their membrane binding mode and relate the orientational freedom to the sterol-dependent tightness of lipid packing. Our results strongly indicate that the membrane insertion of AmB proceeds virtually to completion independent of membrane type, and hence the higher toxicity against fungal membranes may rather result from differences in subsequent oligomerization in the membrane and assembly of monomers into functional transmembrane pores. In particular, the latter could be facilitated by sterol-induced ordering of AmB molecules along the membrane normal, revealed by our free energy profiles. Moreover--in contrast to certain claims--we find no stable binding mode corresponding to the horizontal adsorption of AmB on the membrane surface.

Ceramide–lipid interactions studied by MD simulations and solid-state NMR

Ceramides play a key modulatory role in many cellular processes, which results from their effect on the structure and dynamics of biological membranes. In this study, we investigate the influence of C16-ceramide (C16) on the biophysical properties of DMPC lipid bilayers using solid-state NMR and atomistic molecular dynamics (MD) simulations. MD simulations and NMR measurements were carried out for a pure DMPC bilayer and for a 20% DMPC–C16 mixture. Calculated key structural properties, namely area per lipid, chain order parameters, and mass density profiles, indicate that C16 has an ordering effect on the DMPC bilayer. Furthermore, the simulations predict that specific hydrogen-bonds form between DMPC and C16 molecules. Multi-nuclear solid-state NMR was used to verify these theoretical predictions. Chain order parameters extracted from 13C1H dipole couplings were measured for both lipid and ceramide and follow the trend suggested by the MD simulations. Furthermore, 1H-MAS NMR experiments showed a direct contact between ceramide and lipids.

Exploring Protein-Lipid Interactions using Gramicidin a as a Model System

Gramicidin A (gA) is a 15 amino acid peptide with an alternating L and D sequence that dimerizes to form a membrane-spanning monovalent cation channel. Among gA's key features are four tryptophan (Trp) residues per monomer, which have been implicated as important contributors to the stability and orientation of the channels in bilayers. using molecular dynamics simulations, we have explored the Trp residues' effects on gA channel-lipid interactions. First, a modified gA (Me-gA) was built using 1-methyltryptophan, which disrupts the ability of the indoles to be hydrogen bond donors. These Me-gA dimers were simulated for 100 ns in pure DLPC, DMPC, DOPC, and POPC bilayers, and the results were compared to the previously published results using wild type gA channels. Second, both gA and Me-gA channels were simulated for about 3 μs in bilayer systems with the same average hydrophobic length (equimolar DC16:1PC+DC24:1PC mixture, equimolar DC18:1PC+DC22:1PC mixture, and pure DC20:1PC) to explore the effects of hydrophobic mismatch on lipid distribution adjacent to the protein. In all systems, protein structure and orientation characteristics were calculated. Lipid bilayer properties, such as thickness, surface area per lipid, and lateral compressibility were also quantified as functions of distance from the channel/lipid boundary to elucidate the mutual protein-lipid/ protein-bilayer interactions. Preliminary results show that Me-gA systems exhibit a reduced first shell trough (by 1-3 A) and the bilayers become thicker around Me-gA in all pure bilayer types in comparison to wild type gA. This suggests that the lipids stretch to accommodate the slightly longer hydrophobic thickness of Me-gA. The results from the mixed bilayer simulations indicate lipid redistribution around the channels based on hydrophobic mismatch. These redistribution events appear to begin about 500 ns after beginning the simulations.

Enhanced Lipid Diffusion and Mixing in Accelerated Molecular Dynamics

Accelerated molecular dynamics (aMD) is an enhanced sampling technique that expedites conformational space sampling by reducing the barriers separating various low-energy states of a system. Here, we present the first application of the aMD method on lipid membranes. Altogether, ∼1.5 μs simulations were performed on three systems: a pure POPC bilayer, a pure DMPC bilayer, and a mixed POPC:DMPC bilayer. Overall, the aMD simulations are found to produce significant speedup in trans–gauche isomerization and lipid lateral diffusion versus those in conventional MD (cMD) simulations. Further comparison of a 70-ns aMD run and a 300-ns cMD run of the mixed POPC:DMPC bilayer shows that the two simulations yield similar lipid mixing behaviors, with aMD generating a 2–3-fold speedup compared to cMD. Our results demonstrate that the aMD method is an efficient approach for the study of bilayer structural and dynamic properties. On the basis of simulations of the three bilayer systems, we also discuss the impact of aMD parameters on various lipid properties, which can be used as a guideline for future aMD simulations of membrane systems.

Electric Field Driven Changes of a Gramicidin Containing Lipid Bilayer Supported on a Au(111) Surface

Langmuir-Blodgett and Langmuir-Schaeffer methods were employed to deposit a mixed bilayer consisting of 90% of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 10% of gramicidin (GD), a short 15 residue ion channel forming peptide, onto a Au(111) electrode surface. This architecture allowed us to investigate the effect of the electrostatic potential applied to the electrode on the orientation and conformation of DMPC molecules in the bilayer containing the ion channel. The charge density data were determined from chronocoulometry experiments. The electric field and the potential across the membrane were determined through the use of charge density curves. The magnitudes of potentials across the gold-supported biomimetic membrane were comparable to the transmembrane potential acting on a natural membrane. The information regarding the orientation and conformation of DMPC and GD molecules in the bilayer was obtained from photon polarization modulation infrared reflection absorption spectroscopy (PMIRRAS) measurements. The results show that the bilayer is adsorbed, in direct contact with the metal surface, when the potential across the interface is more positive than -0.4 V and is lifted from the gold surface when the potential across the interface is more negative than -0.4 V. This change in the state of the bilayer has a significant impact on the orientation and conformation of the phospholipid and gramicidin molecules. The potential induced changes in the membrane containing peptide were compared to the changes in the structure of the pure DMPC bilayer determined in earlier studies.

Atomistic Simulation of Hydrophobic Matching Effects on Lipid Composition near a Helical Peptide Embedded in Mixed-Lipid Bilayers

The local lipid composition near a transmembrane helical peptide in mixed-lipid bilayers has been studied using a mixed molecular dynamics (MD) and configuration-bias Monte Carlo method that allows the lateral distribution of lipids to equilibrate much more quickly than is possible by diffusive mixing alone. Gramicidin-A peptide was embedded in bilayer mixtures of DMPC with either DDPC (shorter by four carbons per tail) or DSPC (longer by four carbons per tail) at 330 K to investigate the possibility of lipid sorting by tail length. Conventional MD simulations showed local thickening of the bilayer near the peptide in pure DDPC and local thinning of the bilayer in pure DSPC, with comparatively little perturbation to the thickness of pure DMPC bilayers, suggesting that DMPC has the best matched tail length to the peptide of these three. In 1:1 DMPC:DDPC mixtures, the DMPC lipid was weakly enriched (by about 5%) near the peptide, while in DSPC:DMPC mixtures, no consistent trend was observed. The results underscore the weakness of the coupling between membrane deformation and local composition fluctuations in the absence of spontaneous phase separation.

Solid‐state NMR and simulation studies of equinatoxin II N‐terminus interaction with lipid bilayers

The interaction with model membranes of a peptide, EqtII(1-32), corresponding to the N-terminal region of the pore-forming toxin equinatoxin II (EqtII) has been studied using solid-state NMR and molecular dynamics (MD) simulations. The distances between specifically labeled nuclei in [(19)F-para]Phe16-[1-(13)C]Leu19 and [(19)F-para]Phe16-[(15)N]Leu23 analogs of EqtII(1-32) measured by REDOR in lyophilized peptide were in agreement with published crystal and solution structures. However, in both DMPC and mixed DMPC:SM membrane environments, significant changes in the distances between the labeled amino acid pairs were observed, suggesting changes in helical content around the experimentally studied region, 16-23, in the presence of bilayers. (19)F-(31)P REDOR experiments indicated that the aromatic ring of Phe16 is in contact with lipid headgroups in both membrane environments. For the DMPC:SM mixed bilayers, a closer interaction between Phe16 side chains and lipid headgroups was observed, but an increase in distances was observed for both labeled amino acid pairs compared with those measured for EqtII(1-32) in pure DMPC bilayers. The observed differences between DMPC and DMPC:SM bilayers may be due to the greater affinity of EqtII for the latter. MD simulations of EqtII(1-32) in water, on a pure DMPC bilayer, and on a mixed DMPC:SM bilayer indicate significant peptide secondary structural differences in the different environments, with the DMPC-bound peptide adopting helical formations at residues 16-24, whereas the DMPC:SM-bound peptide exhibits a longer helical stretch, which may contribute to its enhanced activity against PC:SM compared with pure PC bilayers. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Voltammetric study of gold-supported lipid membranes in the presence of perfluorooctanesulphonic acid

The effect of perfluorooctanesulphonic acid (PFOS) on lipid membranes was studied using supported 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) bilayer as the model membrane. Phospholipid bilayer was deposited on gold electrode using a combination of the Langmuir–Blodgett and Langmuir–Schaefer (LB/LS) techniques. Electrodes were modified with two different types of membranes: DMPC bilayers initially containing PFOS and pure DMPC bilayers later exposed to the PFOS solutions. Such approach allowed studying both the changes in membrane characteristic imposed by the perfluorinated compound present in the model membrane and the process of its incorporation into the membrane. Studies with anticancer drug doxorubicin revealed that PFOS inhibits drug transport through the phospholipid bilayer and its effect can be compared to that of cholesterol. Moreover, the different trends observed in the changes in electron transfer rate constant ( k s) calculated for ferricyanides and in peak current of hexaamineruthenium chloride showed that electrostatic interactions between electroactive probes and PFOS molecules incorporating into phospholipid bilayers play an important role and should be taken into account while explaining the interactions of perfluorooctanesulphonic acid with model biological membranes.

Polarization Modulation Infrared Reflection−Absorption Spectroscopy Studies of the Influence of Perfluorinated Compounds on the Properties of a Model Biological Membrane

A combination of the Langmuir-Blodgett and Langmuir-Schaefer techniques has been used to build a 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) bilayer at a Au(111) electrode surface with hydrogen-substituted acyl chains in the top leaflet (solution side) and deuterium-substituted acyl chains in the bottom leaflet (gold side). Polarization modulation infrared reflection-absorption spectroscopy was used to determine changes in the conformation and orientation of the acyl chains of DMPC caused by the incorporation of two selected perfluorinated compounds, perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), into the top leaflet of the bilayer. The incorporation of perfluorinated compounds into the DMPC bilayer caused a broadening of the methylene peaks and a shift in the methylene band positions toward higher frequencies. In addition, the tilt angle of the acyl chains decreased in comparison to the tilt angle of a pure DMPC bilayer. The reported tilt angles were smaller upon insertion of PFOS ( approximately 24 degrees ) than in the presence of PFOA ( approximately 30 degrees ). Overall, the results show that the incorporation of the perfluorinated acids has an effect on the bilayer similar to that of cholesterol by increasing the membrane fluidity and thickness due to a decrease in the tilt angle of the acyl chains.

Non-polar interactions between cholesterol and phospholipids: a molecular dynamics simulation study

A 15-ns molecular dynamics simulation of the fully hydrated dimyristoylphosphatidylcholine-cholesterol (DMPC-Chol) bilayer containing ∼22 mol% Chol was carried out. An 8-ns trajectory was analysed to investigate the effect of Chol on the chain packing in the bilayer core. While the packing of DMPC chains on the smooth α-face side of the Chol ring is similar to that in the pure DMPC bilayer, the packing on the rough β-face side is less regular and less tight. Two methyl groups located on the Chol β-face disturb the packing; in effect, van der Waals (vdW) interactions between Chol rings and DMPC chains are weaker than the ones between sole DMPC chains. VdW interactions between an alkyl chain of DMPC and an isooctyl tail of Chol are similarly strong as those between two DMPC chains.

Non-polar interactions between cholesterol and phospholipids: a molecular dynamics simulation study

A 15-ns molecular dynamics simulation of the fully hydrated dimyristoylphosphatidylcholine-cholesterol (DMPC-Chol) bilayer containing ∼22 mol% Chol was carried out. An 8-ns trajectory was analysed to investigate the effect of Chol on the chain packing in the bilayer core. While the packing of DMPC chains on the smooth α-face side of the Chol ring is similar to that in the pure DMPC bilayer, the packing on the rough β-face side is less regular and less tight. Two methyl groups located on the Chol β-face disturb the packing; in effect, van der Waals (vdW) interactions between Chol rings and DMPC chains are weaker than the ones between sole DMPC chains. VdW interactions between an alkyl chain of DMPC and an isooctyl tail of Chol are similarly strong as those between two DMPC chains.

Effects of Epicholesterol on the Phosphatidylcholine Bilayer: A Molecular Simulation Study

Epicholesterol (Echol) is an epimeric form of cholesterol (Chol). A molecular dynamics simulation of the fully hydrated dimyristoylphosphatidylcholine-Echol (DMPC-Echol) bilayer membrane containing ∼22 mol % of Echol was carried out for 5 ns. A 3-ns trajectory generated between 2 and 5 ns of molecular dynamics simulation was used for analyses to determine the effects of Echol on the membrane properties. As reference systems, pure DMPC and mixed DMPC-Chol bilayers were used. The study shows that Echol, like Chol, changes the organization of the bilayer/water interface and increases membrane order and condensation, but to a lesser degree. Effects of both sterols are based on the same atomic level mechanisms; their different strength arises from different vertical localizations of Echol and Chol hydroxyl groups in the membrane/water interface.

Cholesterol Effects on the Phosphatidylcholine Bilayer Nonpolar Region: A Molecular Simulation Study

A 15-ns molecular dynamics (MD) simulation of the fully hydrated dimyristoylphosphatidylcholine-cholesterol (DMPC-Chol) bilayer in the liquid-crystalline state was carried out to investigate the effect of Chol on the hydrocarbon chain region of the bilayer. The last 8-ns fragment of the generated trajectory was used for analyses. As a reference system, a pure DMPC bilayer (M. Pasenkiewicz-Gierula, Y. Takaoka, H. Miyagawa, K. Kitamura, and A. Kusumi, 1999, Biophys. J. 76:1228–1240) simulated for 14 ns was used. The study shows that a Chol-induced increase of the bulk molecular order parameter along both β- and γ-chain is mainly caused by a decrease of the average tilt of the chains, because the bulk average number of gauche rotamers/myristoyl chain is not significantly changed by Chol. Nevertheless, for DMPCs located near Chol molecules both the number of gauche rotamers/chain and the chain tilt are decreased. The magnitude of the Chol effect on the PC alkyl chains depends, in addition to the PC-Chol distance, on the side of the Chol molecule ( α- or β-face) that the chains are in contact with. This study provides some new insight into the properties of the coexistence region of the partial phase diagram for DMPC-Chol bilayers.

A structural and kinetic study by energy dispersion X-ray diffraction: interaction between 1,4-dihydropyridines and biological membranes

Application of energy dispersive X-ray diffraction to the study of the main transition phase of the pure and doped DMPC ( l α-dimyristoylphosphatidylcholine; 1,2-ditetradecanoyl- sn-glycero-3-phosphatidylcholine) bilayers is reported. The aim is to provide an investigative tool to follow the structural variation induced in these systems by temperature and by insertion of doping substances. Hydrophobic compounds such as 1,4-dihydropyridines are used as doping molecules; Nifedipine and Lacidipine show different physical–chemical properties, modifying in a different way the membrane bilayer and its main transition phase. The results show that this technique is useful for studying these kinds of systems (membrane bilayer or biopolymer), giving information on structural variations that occur in a relatively short time.

Solutes in small amounts provide for lipid-bilayer softness: cholesterol, short-chain lipids, and bola lipids

The effect of the incorporation of small amounts (∼1 mole%) of amphiphilic solutes, such as cholesterol, a short-chain lipid (DC10PC), and a bola lipid, into multilamellar DMPC bilayers is studied by small-angle neutron scattering and differential-scanning calorimetry. The anomalous swelling behavior observed in the transition region of pure DMPC bilayers is interpreted as an indication of bilayer softening and thermally reduced bending rigidity. Small amounts of the solutes are found to maintain or even enhance the bilayer softness. In the case of cholesterol, a systematic study shows that the well-known rigidification effect is observed only for cholesterol concentrations above 3–4 mole%. The results are discussed in relation to the physical properties of internal cell membranes.

Comparison of the Structures of Dimyristoylphosphatidylcholine in the Presence and Absence of Cholesterol by Molecular Dynamics Simulations

Molecular dynamics simulations were carried out on bilayers consisting of dimyristoylphosphatidylcholine dihydrate (DMPC) and DMPC−cholesterol at a 1:1 mole ratio. The calculations were carried out under NPT conditions (constant number of molecules, pressure and temperature, with volume and bilayer area per molecule free to change). Raising the simulation temperature from 200 to 325 K caused the surface area per DMPC molecule to increase from 39 to 50 Å2 in the pure bilayer; the area per heterodimer of DMPC and cholesterol increased from 79 to 91 Å2 for the same temperature range. The properties of the high-temperature state approximates that of a liquid crystalline array, although the area per molecule in the pure DMPC bilayer remains smaller than the experimentally determined value of 65−69 Å2. The number of gauche bonds, the P−N vector angle distribution, and the internal dipole potential are also reported for the simulated liquid crystalline state in the presence and absence of cholesterol.

Interaction of myelin basic protein with single bilayers on a solid support: an NMR, DSC and polarized infrared ATR study

The interaction of myelin basic protein (MBP) with single bilayers on a solid support (planar and spherical support) is studied by deuterium nuclear magnetic resonance ( 2H-NMR), differential scanning calorimetry (DSC) and polarized attenuated total reflection infrared spectroscopy (ATR-IR). The single bilayer consisted of either DMPC or of a binary mixture of DMPC with 10–20 mol% of an acidic phospholipid (DMPG, DMPS or DMPA). All methods applied indicate that MBP strongly interacts with the binary lipid systems but not with the pure DMPC bilayers. The interaction is predominantly electrostatic in nature and does not depend on the choice of a particular acidic lipid (for the binary systems). In particular, the results give no indication for a hydrophobic interaction of MBP with the membrane. Our data provide evidence that, in contrast to previous findings, no demixing and/or domain formation in the binary systems is induced due to the MBP coupling. The infrared order parameter was determined for both lipid components of the binary systems and shows a remarkable change for both lipids due to the interaction with MBP while the NMR order parameter remained essentially unchanged. This is discussed in terms of the different timescales characteristic for both methods. The single supported bilayer responds to the MBP coupling as a whole although only 50% of the bilayer surface is accessible to the protein, indicating a strong coupling between the two bilayer leaflets via the hydrophobic chain region. Moreover, the asymmetric coupling of MBP to the single supported bilayer does not result in a significant redistribution of lipids between the two bilayer leaflets. NMR relaxation time measurements in the headgroup and chain region of DMPG and DMPC suggest that the lateral diffusion coefficient of the acidic lipid decreases significantly due to the coupling with MBP while the zwitterionic DMPC is not affected.

A photographic method of spectrophotometry in the red and infra-red

The effect of perfluorooctanesulphonic acid (PFOS) on lipid membranes was studied using supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayer as the model membrane. Phospholipid bilayer was deposited on gold electrode using a combination of the Langmuir–Blodgett and Langmuir–Schaefer (LB/LS) techniques. Electrodes were modified with two different types of membranes: DMPC bilayers initially containing PFOS and pure DMPC bilayers later exposed to the PFOS solutions. Such approach allowed studying both the changes in membrane characteristic imposed by the perfluorinated compound present in the model membrane and the process of its incorporation into the membrane. Studies with anticancer drug doxorubicin revealed that PFOS inhibits drug transport through the phospholipid bilayer and its effect can be compared to that of cholesterol. Moreover, the different trends observed in the changes in electron transfer rate constant (ks) calculated for ferricyanides and in peak current of hexaamineruthenium chloride showed that electrostatic interactions between electroactive probes and PFOS molecules incorporating into phospholipid bilayers play an important role and should be taken into account while explaining the interactions of perfluorooctanesulphonic acid with model biological membranes.