Phosphatidylinositol induces fluid phase formation and packing defects in phosphatidylcholine model membranes

Liposomes consisted of phosphatidylinositol (PI) and phosphatidylcholine (PC) have been utilized as delivery vehicle for drugs and proteins. In the present work, we studied the effect of soy PI on physical properties of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes such as phase state of lipid bilayer, lipid packing and phase properties using multiple orthogonal biophysical techniques. The 6-dodecanoyl-2-dimethylamino naphthalene (Laurdan) fluorescence studies showed that presence of PI induces the formation of fluid phases in DMPC. Differential scanning calorimetry (DSC), temperature dependent fluorescence anisotropy measurements, and generalized polarization values for Laurdan showed that the presence of as low as 10mol% of PI induces substantial broadening and shift to lower temperature of phase transition of DMPC. The fluorescence emission intensity of DPH labeled, PI containing DMPC lipid bilayer decreased possibly due to deeper penetration of water molecules in lipid bilayer. In order to further delineate the effect of PI on the physico chemical properties of DMPC is due to either significant hydrophobic mismatch between the acyl chains of the DMPC and that of soy PI or due to the inositol head group, we systematically replaced soy PI with PC species of similar acyl chain composition (DPPC and 18:2 (Cis) PC) or with diacylglycerol (DAG), respectively. The anisotropy of PC membrane containing soy PI showed largest fluidity change compared to other compositions. The data suggests that addition of PI alters structure and dynamics of DMPC bilayer in that it promotes deeper water penetration in the bilayer, induces fluid phase characteristics and causes lipid packing defects that involve its inositol head group.     

The interaction of n-alkanols with lipid bilayer membranes: a 2H-NMR study

The interaction of eight n-alkanols with bilayers of dimyristoylphosphatidylcholine (DMPC) has been studied by deuterium nuclear magnetic resonance (2H-NMR). At comparable temperatures and concentrations of solute in the bilayer, order parameters measured at the 1-methylene segment of the n-alkanols show a maximum for n-dodecanol. For both n-dodecanol and n-tetradecanol, orientational ordering shows a maximum at the C-4 to C-7 methylene segments, with labels at both ends of the n-alkanol exhibiting reduced order. These observations are consistent with earlier findings for n-octanol and n-decanol. Unlike the longer chain n-alkanols, ordering in n-butanol decreases from the hydroxyl group end to the methyl group end of the molecule. Orientational ordering at nine inequivalent sites in DMPC, has also been measured as a function of temperature, for bilayers containing n-butanol, n-octanol, n-dodecanol and n-tetradecanol. At the 3R,S sites on the glycerol backbone, for comparable temperatures and solute concentrations, n-butanol produces a larger disordering than the other n-alkanols. This result probably reflects the greater fraction of time spent by the hydroxyl group of n-butanol in the vicinity of the lipid polar head group compared with the hydroxyl group in longer chain n-alkanols. It was found that n-octanol orders the acyl chains of DMPC, unlike n-butanol which disorders them, and the longer chain n-alkanols which have little effect. Within experimental error, the effect of n-dodecanol on order at all sites in DMPC is the same as n-tetradecanol. The influence of n-alkanols on DMPC ordering at twelve sites has been compared with that of cholesterol which is shown to interact with DMPC bilayers in a distinctly different manner from the n-alkanols.     

Effects of lindane on membrane fluidity: intramolecular excimerization of a pyrene derivative and polarization of diphenylhexatriene.

Fluorescence polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH) have been compared with the excimer/monomer fluorescence intensity ratio (I'/I) of 1,3-di(2-pyrenyl)propane, (2Py(3)2Py). This ratio permits evaluation of changes in fluidity of the outer regions of the bilayer, where 2Py(3)2Py preferentially distributes. On the other hand, fluorescence polarization of DPH reports the structural order of the bilayer core. In the fluid phase of DMPC bilayers, for lindane concentrations higher than 25 microM, the excimer/monomer fluorescence intensity ratio (I'/I) decreases, thus reflecting an order increase of the probe environment. However, in the same conditions, the fluorescence polarization of DPH is almost insensitive to any perturbation. Identical results have been obtained in other pure lipid bilayers, namely DPPC and DSPC. However, both probes detect disordering effects of lindane in the gel phase of these lipids. The pyrene probe, unlike DPH, is very sensitive to the pretransitions of DPPC and DSPC, removed in the presence of lindane. Both probes fail to detect any apparent effect of lindane in DMPC bilayers enriched with high cholesterol content (greater than 30 mol%). However, in DMPC bilayers with low cholesterol content (less than 30 mol%), for temperatures below the phase transition of DMPC, both probes detect fluidizing effects induced by lindane. Nevertheless, above the phase transition of DMPC, 2Py(3)2Py detects ordering effects of lindane, whereas DPH detects hardly any effect. These results in DMPC bilayers with low cholesterol content are qualitatively similar to those described for DMPC without cholesterol.     

Effects of phospholipid unsaturation on the bilayer nonpolar region: a molecular simulation study

Molecular dynamics simulations of two monounsaturated phosphatidylcholine (PC) bilayers made of 1-palmitoyl-2-oleoyl-PC (POPC; cis-unsaturated) and 1-palmitoyl-2-elaidoyl-PC (PEPC; trans-unsaturated) were carried out to investigate the effect of a double bond in the PC beta-chain and its conformation on the bilayer core. Four nanosecond trajectories were used for analyses. A fully saturated 1,2-dimyristoyl-PC (DMPC) bilayer was used as a reference system. In agreement with experimental data, this study shows that properties of the PEPC bilayer are more similar to those of the DMPC than to the POPC bilayer. The differences between POPC and PEPC bilayers may be attributed to the different ranges of angles covered by the torsion angles beta10 and beta12 of the single bonds next to the double bond in the oleoyl (O) and elaidoyl (E) chains. Broader distributions of beta10 and beta12 in the E chain than in the O chain make the E chain more flexible. In effect, the packing of chains in the PEPC bilayer is similar to that in the DMPC bilayer, whereas that in the POPC bilayer is looser than that in the DMPC bilayer. The effect of the cis-double bond on torsions at the beginning of the O chain (beta4 and beta5) is similar to that of cholesterol on these torsions in a myristoyl chain.     

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 approximately 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 alpha-face side of the Chol ring is similar to that in the pure DMPC bilayer, the packing on the rough beta-face side is less regular and less tight. Two methyl groups located on the Chol beta-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.     

The orientation effect of gramicidin A on bicelles and Eu3+ -doped bicelles as studied by solid-state NMR and FT-IR spectroscopy

We have explored the effect of gramicidin A (gA) on bicelle (Bic) orientation in the absence and presence of Eu(3+) by (31)P and (2)H NMR at different DMPC/gA ratios. FT-IR spectroscopy was used to assess the lipid chain ordering and verify the transmembrane peptide conformation. Our results show a time-dependent flipping of the bilayer normal alignment at high temperatures and high proportion of gA. The results are explained by both the diamagnetic susceptibility anisotropy of the beta(6.3) helical peptides and viscosity of the lipid mixture. The concentration effect of gramicidin on Bic/Eu(3+) is compared to that on Eu(3+)-doped DMPC liposomes. The Bic/Eu(3+) system is no longer oriented in the presence of gA and adopts a vesicular morphology while the peptide incorporation induces the formation of ellipsoidal DMPC/Eu(3+) assemblies aligned with their normal parallel to the magnetic field. The difference is explained in terms of lipid chain disorder and size of the bilayers.     

Effects of pH-induced variations of the charge of the transmembrane alpha-helical peptide Ac-K2(LA)12K2-amide on the organization and dynamics of the host dimyristoylphosphatidylcholine bilayer membrane

The effects of the transmembrane alpha-helical peptide Ac-K(2)(LA)(12)K(2)-amide ((LA)(12)) on the phase transition and dynamics of saturated dimyristoylphosphatidylcholine (DMPC) membranes were investigated at different pH using conventional and saturation-recovery EPR observations of phosphatidylcholine spin labels. At a peptide-to-DMPC ratio of 1/10, the main phase-transition temperature of the DMPC bilayer is decreased by 4.0 degrees C when measured at pH 7.0, by 1.6 degrees C when measured at pH 9.5, and not affected when measured at pH 11.5. This reversible pH effect is due to the subsequent neutralization of the positive charges of lysine side chains at both ends of (LA)(12). Apparent pK(a)s of the lysine side chain amino groups of (LA)(12) in DMPC bilayer are 8.6 and approximately 10.9, as compared with the pK(a) value of 10.5 for these groups when lysine is dissolved in water. Saturation-recovery curves as a function of oxygen concentration using phosphatidylcholine spin labels in DMPC bilayer containing (LA)(12) are always mono-exponential when measured at pH 7.0 and 9.5. This observation is consistent with the hypothesis that the lipid exchange rates among the bulk, boundary, and (LA)(12)-rich regions are faster than 0.5 micros, the electron spin-lattice relaxation time in the presence of molecular oxygen, suggesting that stable oligomers of (LA)(12) do not form. Neutralization of one lysine side chain positive charge on each end of the peptide significantly decreases the ordering effect of (LA)(12) on the lipid hydrocarbon chains, while its effect on the reorientational motion of terminal groups of lipid hydrocarbon chains is rather moderate. It does not affect the local diffusion-solubility product of oxygen measured in the DMPC-(LA)(12) membrane interior.     

Molecular dynamics simulations of local anesthetic articaine in a lipid bilayer

In order to investigate structural and dynamical properties of local anesthetic articaine in a model lipid bilayer, a series of molecular dynamics simulations have been performed. Simulations were carried out for neutral and charged (protonated) forms of articaine inserted in fully hydrated dimyristoylphosphatidylcholine (DMPC) lipid bilayer. For comparison purpose, a fully hydrated DMPC bilayer without articaine was also simulated. The length of each simulation was 200 ns. Various properties of the lipid bilayer systems in the presence of both charged and uncharged forms of articaine taken at two different concentrations have been examined: membrane area per lipid, mass density distributions, order parameters, radial distribution functions, head group tilt, diffusion coefficients, electrostatic potential, etc, and compared with results of previous simulations of DMPC bilayer in the presence of lidocaine. It was shown that addition of both charged and neutral forms of articaine causes increase of the dipole electrostatic potential in the membrane interior.     

Atomic detail peptide-membrane interactions: molecular dynamics simulation of gramicidin S in a DMPC bilayer

Molecular dynamics simulations have been performed of the sequence-symmetric cyclic decapeptide antibiotic gramicidin S (GS), in interaction with a hydrated dimyristoylphosphatidylcholine (DMPC) bilayer, and the results compared with a "control" simulation of the system in the absence of GS. Following experimental evidence, the GS was initially set in a single antiparallel beta-sheet conformation with two Type II' beta-turns in an amphiphilic interaction with the membrane. This conformation and position remained in the 6.5 ns simulation. Main-chain dihedrals are on average approximately 26 degrees from those determined by NMR experiment on GS in dimethylsulfoxide (DMSO) solution. Sequence-symmetric main-chain and side-chain dihedral angle pairs converge to within approximately 5 degrees and approximately 10 degrees, respectively. The area per lipid, lipid tail order parameters, and quadrupole spin-lattice relaxation times of the control simulation are mostly in good agreement with corresponding experiments. The GS has little effect on the membrane dipole potential or water permeability. However, it is found to have a disordering effect (in agreement with experiment) and a fluidifying effect on lipids directly interacting with it, and an ordering effect on those not directly interacting.     

Spin-label electron spin resonance studies on the mode of anchoring and vertical location of the N-acyl chain in N-acylphosphatidylethanolamines

Electron spin resonance (ESR) studies have been performed on N-myristoyl dimyristoylphosphatidylethanolamine (N-14-DMPE) membranes using both phosphatidylcholines spin-labeled at different positions in the sn-2 acyl chain and N-acyl phosphatidylethanolamines spin-labeled in the N-acyl chain to characterize the location and mobility of the N-acyl chain in the lipid membranes. Comparison of the positional dependences of the spectral data for the two series of spin-labeled lipids suggests that the N-acyl chain is positioned at approximately the same level as the sn-2 chain of the phosphatidylcholine spin-label. Further, similar conclusions are reached when the ESR spectra of the N-acyl PE spin-labels in dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylethanolamine (DMPE) host matrixes are compared with those of phosphatidylcholine spin-labels in these two lipids. Finally, the chain ordering effect of cholesterol has also been found to be similar for the N-acyl PE spin-label and PC spin-labels, when the host matrix is either DMPC and cholesterol or N-14-DMPE and cholesterol at a 6:4 mole ratio. In both cases, the gel-to-liquid crystalline phase transition is completely abolished but cholesterol perturbs the gel-phase mobility of N-14-DMPE more readily than that of DMPC. These results demonstrate that the long N-acyl chains are anchored firmly in the hydrophobic interior of the membrane, in an orientation that is parallel to that of the O-acyl chains, and are located at nearly the same vertical position as that of the sn-2 acyl chains in the lipid bilayer. There is a high degree of dynamic compatibility between the N-acyl chains and the O-acyl chains of the lipid bilayer core, although bilayers of N-acyl phosphatidylethanolamines possess a more hydrophobic interior than phosphatidylcholine bilayers. These results provide a structural basis for rationalizing the biological properties of NAPEs.     

Amphotericin and model membranes. The effect of amphotericin B on cholesterol-containing systems as viewed by 2H-NMR

The interactions between amphotericin B and sterol-containing model membranes were monitored by 2H-NMR of deuterium-labelled dimyristoylphosphatidylcholine (DMPC), cholesterol and epicholesterol. The addition of amphotericin B to a cholesterol/DMPC (3:7) system was perceived differently by the lipid, depending upon the depth in the bilayer: no structural change was manifest in the acyl chain region associated with the plateau in molecular ordering (C4'), whereas the lipid clearly senses two environments near the center of the bilayer (C13', C14'). The amount as well as the ordering properties of the more ordered antibiotic-induced component, sensed at C14', increased with decreasing temperature. The structural parameters of deuterium-labelled cholesterol in cholesterol/DMPC mixtures were unchanged upon addition of amphotericin B, regardless of the bilayer depth. Upon addition of amphotericin B, the lipid T1 values are unchanged, whereas the T2 values are reduced by a factor of 2. The minimum in T1 observed for cholesterol in DMPC at 32-35 degrees C was shifted towards 38-40 degrees C in the presence of amphotericin B. Epicholesterol-containing dispersions of DMPC had properties similar to those of their cholesterol-containing analogs; a noticeable difference between the systems was an approx. 10% increase in the segmental order parameters on the addition of amphotericin B to the system containing the alpha-isomer of cholesterol. The concept of a dynamic complexation between amphotericin B and sterol is discussed.     

Structural divergence among cannabinoids influences membrane dynamics: a 2H solid-state NMR analysis

Cannabinoids are compounds that can modulate neuronal functions and immune responses via their activity at the CB(1) receptor. We used (2)H NMR order parameters and relaxation rate determination to delineate the behavior of magnetically aligned phospholipid bilayers in the presence of several structurally distinct cannabinoid ligands. THC (Delta(9)-Tetrahydrocannabinol) and WIN-55,212-2 were found to lower the phase transition temperature of the DMPC and to destabilize their acyl chains leading to a lower average S(CD) ( approximately 0.13), while methanandamide and CP-55,940 exhibited unusual properties within the lipid bilayer resulting in a greater average S(CD) ( approximately 0.14) at the top of the phospholipid upper chain. The CB(1) antagonist AM281 had average S(CD) values that were higher than the pure DMPC lipids, indicating a stabilization of the lipid bilayer. R(1Z) versus |S(CD)|(2) plots indicated that the membrane fluidity is increased in the presence of THC and WIN-55,212-2. The interaction of CP-55,940 with a variety of zwitterionic and charged membranes was also assessed. The unusual effect of CP-55,940 was present only in bicelles composed of DMPC. These studies strongly suggest that cannabinoid action on the membrane depends upon membrane composition as well as the structure of the cannabinoid ligands.     

Iodide penetration into lipid bilayers as a probe of membrane lipid organization.

The quenching efficiency of iodide as a penetrating fluorescence quencher for a membrane-associated fluorophore was utilized to measure the molecular packing of lipid bilayers. The KI quenching efficiency of tryptophan-fluorescence from melittin incorporated in DMPC bilayer vesicles peaks at the phase transition temperature (24 degrees C) of DMPC, whereas acrylamide quenching efficiency does not depend on temperature. The ability of iodide to penetrate the hydrocarbon region of the bilayer was examined by measuring the fluorescence quenching of the pyrene-phosphatidylcholine incorporated into DMPC vesicles (pyrene was attached to the 10th carbon of the sn-2 chain). The quenching efficiency of pyrene by iodide again shows a maximum at the lipid phase transition. We conclude that iodide penetrates the membrane hydrocarbon region at phase transition through an increased number of bilayer defects. The magnitude of change in quenching efficiency of iodide during lipid phase transition provides a sensitive technique to probe the lipid organization in membranes.     

Investigation of phospholipid area compression induced by calcium-mediated dextran sulfate interaction.

The association of anionic polyelectrolytes such as dextran sulfate (DS) to zwitterionic phospholipid surfaces via Ca(2+) bridges results in a perturbation of lipid packing at physiologically relevant Ca(2+) concentrations. Lipid area compression was investigated in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) multilamellar bilayer dispersions by (2)H-NMR and in monolayer studies. Binding of DS to DMPC surfaces via Ca(2+) results in denser lipid packing, as indicated by higher lipid chain order. DMPC order parameters are homogeneously increased throughout the lipid bilayer. Higher order translates into more extended hydrocarbon chains and decreased average lipid area per molecule. Area compression is reported as a function of DS concentration and molecular weight. Altering the NaCl and Ca(2+) concentrations modified electrostatic interactions between DS and phospholipid. A maximal area reduction of DeltaA = 2.7 A(2) per DMPC molecule is observed. The lipid main-phase transition temperature increases upon formation of DMPC/Ca(2+)/DS-complexes. Lipid area compression after addition of DS and Ca(2+) to the subphase was also observed in monolayer experiments. A decrease in surface tension of up to 3.5 mN/m at constant molecular area was observed. DS binds to the lipid headgroups by formation of Ca(2+) bridges without penetrating the hydrophobic region. We suggest that area compression is the result of an attractive electrostatic interaction between neighboring lipid molecules induced by high local Ca(2+) concentration due to the presence of DS. X-ray diffraction experiments demonstrate that DS binding to apposing bilayers reduces bilayer separation. We speculate that DS binding alters the phase state of low-density lipoproteins that associate with polyelectrolytes of the arterial connective tissue in the early stages of arteriosclerosis.     

Interactions of cholesterol with the membrane lipid matrix. A solid state NMR approach.

The effects of cholesterol on the structure and dynamics of dimyristoylphosphatidylcholine (DMPC) model membranes have been monitored as functions of temperature and cholesterol concentration in the membrane. The use of deuterium labels both on the cholesterol fused ring system and on the lipid chains in conjunction with solid state deuterium nuclear magnetic resonance (2H-NMR) afforded to monitor the degree of ordering of both molecules in a mixed system. The degree of ordering of the lipid head group was followed by phosphorus-31 (31P)-NMR. New findings on the effect of cholesterol on DMPC may be summarized as follows: i) cholesterol disorders the lipid chains below temperature of the DMPC gel-to-fluid transition (Tc) and orders them above; the effect is linear with cholesterol concentration at 0 and 60 degrees C but for intermediate temperatures, a saturation effect is observed at 20-30 mol %; ii) the ordering-disordering effects are perceived similarly by all chain segments with, however, a greater sensitivity for positions near the bilayer center; iii) below Tc, the lipid head group is considerably disordered by increasing amounts of cholesterol but slightly affected above; iv) the degree of ordering of cholesterol is quasi temperature independent for fractions greater than or equal to 30%; v) the average orientation of the cholesterol rigid body is perpendicular to the bilayer surface and exhibits little variations with temperature and cholesterol concentration. Variations in membrane dynamics are interpreted in terms of cholesterol-induced changes in bilayer thickness.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein-lipid interactions. A nuclear magnetic resonance study of sarcoplasmic reticulum Ca2,Mg2+-ATPase, lipophilin, and proteolipid apoprotein-lecithin systems and a comparison with the effects of cholesterol.

Deuterium Fourier transform nuclear magnetic resonance (NMR) spectra at 34 MHz (corresponding to a magnetic field strength of 5.2 T) have been obtained of a variety of protein-lipid systems containing specifically deuterated phospholipids. The following systems were investigated as a function of temperature: sarcoplasmic reticulum ATPase (ATP phosphohydrolase, EC 3.6.1.3) complexed with 1-myristoyl-2-(14,14,14-trideuteriomyristoyl)-sn-glycero-3-phosphocholine (DMPC-d3) or 1,2-bis(16,16,16-trideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-k6); human brain lipophilin complexed with DPPC-d6 or 1,2-bis(6,6-dideuteriopalmitoyl)-sn-glycero-3-phosphocholine (DPPC-6,6-d4); beef brain myelin proteolipid apoprotein (PLA) reconstituted with DMPC labeled as CD2 (or CD3) at one or more of positions 3, 4, 6, 8, 10, 12, or 14 of the sn-2 chain. For purposes of comparison, spectra were also obtained for bilayers containing cholesterol (CHOL). The results show that proteins either disorder or have little effect on hydrocarbon chain order in membranes above the gel to liquid-crystal phase transition temperature (Tc) of the pure lipids. Cholesterol, however, causes a very large ordering of the hydrocarbon chains above Tc, but both cholesterol and protein prevent chain crystallization (by effectively disordering chain packing) immediately below Tc. No evidence for any ordered "boundary lipid" in association with protein was found above Tc, perhaps due to the rough nature of protein surfaces. Above Tc, exchange between free bilayer and protein associated lipid is fast on the time scale of the deuterium NMR experiment (greater than or similar to 10(3) s-1). We have also obtained proton-decoupled phosphorus-31 nuclear magnetic resonance spectra at 60.7 MHz (corresponding to a magnetic field strength of 3.5 T) of DMPC, DMPC-AT-Pase, and DMPC-CHOL complexes. The results indicate that ATPase and CHOL CAUSE SMALL DECREASES IN 31P chemical shielding anisotropies but that in addition ATPase causes a four- to fivefold increase in 31P spin-lattice and Carr-Purcell spin-spin relaxation rates, suggesting the possibility of polar group protein-lipid interaction leading to increased correlation times in the region of the lipid phosphate head group.     

Partitioning and membrane disordering effects of dopamine antagonists: influence of lipid peroxidation, temperature, and drug concentration.

The effect of four dopamine antagonists (spiperone, haloperidol, pimozide, and domperidone) on the lipid order of caudate nucleus microsomal membranes and on liposomes from membrane lipid extracts was evaluated and related to the partition coefficients (Kp) of the drugs. Lipid membrane order was determined by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe of the membrane core and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) as a probe of the membrane surface. Dopamine antagonists decrease the fluorescence polarization of both probes, indicating that they disorder the membrane lipids at different depths. Pimozide and domperidone, the drugs with higher Kp values, are more effective at decreasing the polarization of DPH, a probe of the membrane core, than that of TMA-DPH. In contrast, spiperone and haloperidol, which have lower values for Kp, induce more significant decreases in TMA-DPH depolarization, a probe of the membrane surface. These findings indicate that higher partition coefficients of the drugs are directly correlated with an increase of fluidity in the hydrophobic core of brain membranes. Ascorbate/Fe(2+)-induced membrane lipid peroxidation increases membrane order. Membrane lipid peroxidation decreases the partition coefficients of the dopamine antagonists tested. Increasing temperature (4-37 degrees C) decreases membrane order, but temperature effect is less evident after lipid peroxidation. The disordering effect of dopamine antagonists increases with increasing drug concentrations (1-15 microM), a maximum being observed at 10 microM. However, this effect is also less evident after membrane lipid peroxidation. We can conclude that dopamine antagonists and membrane lipid peroxidation affect membrane lipid order and that the action of these drugs is dependent on initial bilayer fluidity. Membrane lipid peroxidation increases membrane order while dopamine antagonists show a disordering effect of membrane phospholipids. This disordering effect can indirectly influence the activity of membrane proteins and it is one of the mechanisms through which membrane function can be altered by these drugs.     

Spectroscopic characterization of DMPC/DOTAP cationic liposomes and their interactions with DNA and drugs

Gene and synthetic drug-delivery vectors have been developed and characterized to treat several genetic diseases and cancers. Our study aims at characterizing cationic liposomes containing the zwitterionic phospholipid DMPC and the cationic lipid DOTAP as well as their interactions with two types of DNA and a new class of antineoplastic agents derived from arylchloroethylureas (CEU). Results obtained using FTIR spectroscopy as well as ³¹P and ²H NMR indicate that DMPC and DOTAP form cationic liposomes in a highly disordered fluid phase at a molar ratio of 1:1. In addition, the FTIR results indicate that the presence of DNA or CEUs within the liposomes does not significantly affect the conformational order of both the DMPC and DOTAP acyl chains. Our results therefore provide a detailed characterization of complexes between cationic liposomes and both DNA and drugs and indicate that these complexes are stable and fluid assemblies.     

Binding of Aβ peptide creates lipid density depression in DMPC bilayer

Using isobaric–isothermal replica exchange molecular dynamics and all-atom explicit water model we study the impact of Aβ monomer binding on the equilibrium properties of DMPC bilayer. We found that partial insertion of Aβ peptide into the bilayer reduces the density of lipids in the binding “footprint” and indents the bilayer thus creating a lipid density depression. Our simulations also reveal thinning of the bilayer and a decrease in the area per lipid in the proximity of Aβ. Although structural analysis of lipid hydrophobic core detects disordering in the orientations of lipid tails, it also shows surprisingly minor structural perturbations in the tail conformations. Finally, partial insertion of Aβ monomer does not enhance water permeation through the DMPC bilayer and even causes considerable dehydration of the lipid–water interface. Therefore, we conclude that Aβ monomer bound to the DMPC bilayer fails to perturb the bilayer structure in both leaflets. Limited scope of structural perturbations in the DMPC bilayer caused by Aβ monomer may constitute the molecular basis of its low cytotoxicity.     

Dibucaine effects on structural and elastic properties of lipid bilayers

In this work we report the interaction effects of the local anesthetic dibucaine (DBC) with lipid patches in model membranes by Atomic Force Microscopy (AFM). Supported lipid bilayers (egg phosphatidylcholine, EPC and dimyristoylphosphatidylcholine, DMPC) were prepared by fusion of unilamellar vesicles on mica and imaged in aqueous media. The AFM images show irregularly distributed and sized EPC patches on mica. On the other hand DMPC formation presents extensive bilayer regions on top of which multibilayer patches are formed. In the presence of DBC we observed a progressive disruption of these patches, but for DMPC bilayers this process occurred more slowly than for EPC. In both cases, phase images show the formation of small structures on the bilayer surface suggesting an effect on the elastic properties of the bilayers when DBC is present. Dynamic surface tension and dilatational surface elasticity measurements of EPC and DMPC monolayers in the presence of DBC by the pendant drop technique were also performed, in order to elucidate these results. The curve of lipid monolayer elasticity versus DBC concentration, for both EPC and DMPC cases, shows a maximum for the surface elasticity modulus at the same concentration where we observed the disruption of the bilayer by AFM. Our results suggest that changes in the local curvature of the bilayer induced by DBC could explain the anesthetic action in membranes.