Interaction of ceramides with phosphatidylcholine, sphingomyelin and sphingomyelin/cholesterol bilayers

Ceramides (Cers) may exert their biological activity through changes in membrane structure and organization. To understand this mechanism, the effect of Cer on the biophysical properties of phosphatidylcholine, sphingomyelin (SM) and SM/cholesterol bilayers was determined using fluorescence probe techniques. The Cers were bovine brain Cer and synthetic Cers that contained a single acyl chain species. The phospholipids were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glyero-3-phosphocholine (DPPC) and bovine brain, egg yolk and bovine erythrocyte SM. The addition of Cer to POPC and DPPC bilayers that were in the liquid-crystalline phase resulted in a linear increase in acyl chain order and decrease in membrane polarity. The addition of Cer to DPPC and SM bilayers also resulted in a linear increase in the gel to liquid-crystalline phase transition temperature (T(M)). The magnitude of the change was dependent upon Cer lipid composition and was much higher in SM bilayers than DPPC bilayers. The addition of 33 mol% cholesterol essentially eliminated the thermal transition of SM and SM/Cer bilayers. However, there is still a linear increase in acyl chain order induced by the addition of Cer. The results are interpreted as the formation of DPPC/Cer and SM/Cer lipid complexes. SM/Cer lipid complexes have higher T(M)s than the corresponding SM because the addition of Cer reduces the repulsion between the bulky headgroup and allows closer packing of the acyl chains. The biophysical properties of a SM/Cer-rich bilayer are dependent upon the amount of cholesterol present. In a cholesterol-poor membrane, a sphingomyelinase could catalyze the isothermal conversion of a liquid-crystalline SM bilayer to a gel phase SM/Cer complex at physiological temperature.     

Using micropatterned lipid bilayer arrays to measure the effect of membrane composition on merocyanine 540 binding

The lipophilic dye merocyanine 540 (MC540) was used to model small molecule-membrane interactions using micropatterned lipid bilayer arrays (MLBAs) prepared using a 3D Continuous Flow Microspotter (CFM). Fluorescence microscopy was used to monitor MC540 binding to fifteen different bilayer compositions simultaneously. MC540 fluorescence was two times greater for bilayers composed of liquid-crystalline (l.c.) phase lipids (1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) compared to bilayers in the gel phase (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)). The effect cholesterol (CHO) had on MC540 binding to the membrane was found to be dependent on the lipid component; cholesterol decreased MC540 binding in DMPC, DPPC and DSPC bilayers while having little to no effect on the remaining l.c. phase lipids. MC540 fluorescence was also lowered when 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS) was incorporated into DOPC bilayers. The increase in the surface charge density appears to decrease the occurrence of highly fluorescent monomers and increase the formation of weakly fluorescent dimers via electrostatic repulsion. This paper demonstrates that MLBAs are a useful tool for preparing high density reproducible bilayer arrays to study small molecule-membrane interactions in a high-throughput manner.     

Pressure effects on the physical properties of lipid bilayers detected by trans-parinaric acid fluorescence decay.

The effects of hydrostatic pressure on the physical properties of large unilamellar vesicles of single lipids dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) and lipid mixtures of DMPC/DPPC have been studied from time-resolved fluorescence of trans-parinaric acid. Additional experiments were carried out using diphenylhexatriene to compare the results extracted from both probes. Fluorescence decays were analyzed by the maximum entropy method. Pressure does not influence the fluorescence lifetime distribution of trans-parinaric acid in isotropic solvents. However, in pressurized lipid bilayers an abrupt change was observed in the lifetime distribution which was associated with the isothermal pressure-induced phase transition. The pressure to temperature equivalence values, dT/dP, determined from the midpoint of the phase transitions, were 24 and 14.5 degrees C kbar-1 for DMPC and POPC, respectively. Relatively moderate pressures of about 500 bar shifted the DMPC/DPPC phase diagram 11.5 degrees C to higher temperatures. The effects of pressure on the structural properties of these lipid vesicles were investigated from the anisotropy decays of both probes. Order parameters for all systems increased with pressure. In the gel phase of POPC the order parameter was smaller than that obtained in the same phase of saturated phospholipids, suggesting that an efficient packing of the POPC hydrocarbon chains is hindered.     

Differential scanning calorimetric studies of the thermotropic phase behavior of membranes composed of dipalmitoyllecithin and mixed-acid unsaturated lecithins

The lecithins 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) have been synthesized by reacylation of the appropriate lysolecithins with fatty acid anhydrides. These lecithins have been used to make model membranes in mixtures with dipalmitoyllecithin (DPPC), and phase diagrams of the two bilayer systems have been constructed. These diagrams show that there is essentially no gel-state miscibility in the POPC-DPPC bilayers at any composition, and that SOPC-DPPC bilayers show gel-state immiscibility at DPPC concentrations of less than 50 mol%, and partial miscibility above 50 mol% DPPC. Analysis of the POPC-DPPC phase diagram on the assumption of athermal solution in the liquid-crystalline phase shows that the two lipids mix nearly randomly above the phase transition. The liquidus curve of SOPC-DPPC bilayers showed deviations from calculated ideal behaviour, which indicated that there is a small excess tendency for the formation of pairs of like molecules in SOPC-DPPC bilayers in the liquid-crystalline phase. Thus, in the liquid-crystalline phase, SOPC and DPPC do not pack quite as well as do POPC and DPPC.     

Effect of cholesterol on the formation of an interdigitated gel phase in lysophosphatidylcholine and phosphatidylcholine binary mixtures

We previously reported that 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) forms an interdigitated gel phase in the presence of 1-palmitoyl-sn-glycero-3-phosphocholine (16:0LPC) at concentrations below 30 mol%. In the present investigation, fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), X-ray diffraction, and differential scanning calorimetry (DSC) were used to investigate the effect of cholesterol on the phase behavior of 16:0LPC/DPPC binary mixtures. At 25 degrees C, 30 mol% 16:0LPC significantly decreases the DPH fluorescence intensity during the transition of DPPC from the L(beta') phase to the L(betaI) phase. However, the addition of cholesterol to 16:0LPC/DPPC mixtures results in a substantial increase in fluorescence intensity. The changes in DPH fluorescence intensity reflect the probe's redistribution from an orientation parallel to the acyl chain to the center of the bilayer, suggesting a bilayer structure transition from interdigitation to noninterdigitation. The normal repeat period of small angle X-ray diffraction patterns can be restored and a reflection appears at 0.42 nm with a broad shoulder around 0.41 nm in wide angle X-ray diffraction patterns when 10 mol% cholesterol is incorporated into 30 mol% 16:0LPC/DPPC vesicles, indicating that the mixtures are in the gel phase (L(beta')). Moreover, DSC results demonstrate that 10 mol% cholesterol is sufficient to significantly decrease the main enthalpy, cooperativity and lipid chain melting of 30 mol% 16:0LPC/DPPC binary mixtures, which are L(betaI), indicating that the transition of the interdigitated phase is more sensitive to cholesterol than that of the noninterdigitated phase. Our data imply that the interdigitated gel phase induced by 16:0LPC is prevented in the presence of 10 mol% cholesterol, but unlike ethanol, an increasing concentration of 16:0LPC is not able to restore the interdigitation structure of the lipid mixtures.     

The effects of oxidised phospholipids and cholesterol on the biophysical properties of POPC bilayers

Oxidation of unsaturated membrane phospholipids by oxidative stress is associated with inflammation, infection, numerous diseases and neurodegenerative disorders. Lipid oxidation is observed in experimental samples when the parent lipid is exposed to oxidative stressors. The effect of phospholipid oxidation on the properties of biological membranes are still being explored, while low concentrations (0.1–2.0?mol%) of oxidised phospholipids are associated with disease states [1]. Previous computational studies have focused on the effect of high concentrations (~50?mol%) of oxidised phospholipids on binary lipid bilayers. This work systematically characterises the effect of lower concentrations (~10?mol%) of two oxidised lipid species, PoxnoPC (1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine) or PazePC (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine), on POPC/cholesterol and pure POPC bilayers. During μs atomistic simulations in pure POPC bilayers, PoxnoPC and PazePC reoriented their oxidised sn-2 acyl chains towards the solution, and PazePC adopted an extended conformation. The addition of 20?mol% cholesterol not only modulated the fluidity of the bilayers; it also modulated the flexibility of the PoxnoPC oxidised sn-2 tail, reducing bilayer disorder. In contrast, the addition of cholesterol had little effect on bilayers containing PazePC. Our studies show that the effect of oxidised lipids on the biophysical properties of a multicomponent bilayer cannot be intuitively extrapolated from a binary lipid system.     

The effect of cholesterol on measured interaction and compressibility of dipalmitoylphosphatidylcholine bilayers

We have examined the phase diagram of dipalmitoylphosphatidylcholine (DPPC)--cholesterol-water mixtures at low cholesterol content, and report phase separation between 3 and 10 mol% cholesterol. The two lamellar phases at equilibrium in this region appear to be pure DPPC and 11 mol% cholesterol in DPPC. For these two lamellar phases, which are made up of alternating layers of water and bimolecular lipid leaflets, we have measured the forces of interaction between leaflets and the lateral pressure and compressibility of the leaflets. Both bilayers experience a strong repulsive force when forced together only a few ?ngstr?ms (1 A = 0.1 nm) closer than their maximum separation in excess water. However, the presence of 11 mol% cholesterol causes the bilayers to move apart of 35-A separation from the 19-A characteristic of pure DPPC in excess water. This swelling may result from a decrease in van der Waals attraction between bilayers or from an increase in bilayer repulsion. Differences in bilayer interaction can be a cause for phase separation. More importantly these differences can cause changes in the composition of regions of membranes approaching contact. At 11 mol%, cholesterol substantially increases the lateral compressibility of DPPC bilayers leading to higher lateral density fluctuations and potentially higher bilayer permeability.     

Phospholipid packing and hydration in pulmonary surfactant membranes and films as sensed by LAURDAN

The efficiency of pulmonary surfactant to stabilize the respiratory surface depends critically on the ability of surfactant to form highly packed films at the air-liquid interface. In the present study we have compared the packing and hydration properties of lipids in native pulmonary surfactant and in several surfactant models by analyzing the pressure and temperature dependence of the fluorescence emission of the LAURDAN (1-[6-(dimethylamino)-2-naphthyl]dodecan-1-one) probe incorporated into surfactant interfacial films or free-standing membranes. In interfacial films, compression-driven changes in the fluorescence of LAURDAN, evaluated from the generalized polarization function (GPF), correlated with changes in packing monitored by surface pressure. Compression isotherms and GPF profiles of films formed by native surfactant or its organic extract were compared at 25 or 37 °C to those of films made of dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), DPPC/phosphatidylglycerol (PG) (7:3, w/w), or the mixture DPPC/POPC/palmitoyloleoylphosphatidylglycerol (POPG)/cholesterol (Chol) (50:25:15.10), which simulates the lipid composition of surfactant. In general terms, compression of surfactant films at 25 °C leads to LAURDAN GPF values close to those obtained from pure DPPC monolayers, suggesting that compressed surfactant films reach a dehydrated state of the lipid surface, which is similar to that achieved in DPPC monolayers. However, at 37 °C, the highest GPF values were achieved in films made of full surfactant organic extract or the mixture DPPC/POPC/POPG/Chol, suggesting a potentially important role of cholesterol to ensure maximal packing/dehydration under physiological constraints. Native surfactant films reached high pressures at 37 °C while maintaining relatively low GPF, suggesting that the complex three-dimensional structures formed by whole surfactant might withstand the highest pressures without necessarily achieving full dehydration of the lipid environments sensed by LAURDAN. Finally, comparison of the thermotropic profiles of LAURDAN GPF in surfactant model bilayers and monolayers of analogous composition shows that the fluorophore probes an environment that is in average intrinsically more hydrated at the interface than inserted into free-standing bilayers, particularly at 37 °C. This effect suggests that the dependence of membrane and surfactant events on the balance of polar/non-polar interactions could differ in bilayer and monolayer models, and might be affected differently by the access of water molecules to confined or free-standing lipid structures.     

Controlling association of vesicle embedded peptides by alteration of the physical state of the lipid matrix

We report here the reversible association of a designed peptide embedded in a lipid membrane through a stimulus-sensitive trigger that changes the physical state of the bilayer matrix. A peptide designed with the classical 4-3 heptad repeat of coiled coils, equipped with leucine residues at all canonical interface positions, TH1, was rendered membrane soluble by replacement of all exterior residues with randomly selected hydrophobic amino acids. Insertion of TH1 into large unilamellar phosphatidylcholine vesicles was followed by monitoring tryptophan fluorescence. Peptide insertion was observed when the lipids were in the liquid-crystalline state [1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)] but not when they were in the crystalline phase [1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)]. Formation of a trimeric alpha-helical bundle in lipid bilayers was followed by fluorescence resonance energy transfer. Global fit analysis revealed a monomer--trimer equilibrium with a dissociation constant of around 10(-5) [corrected] MF(2). A lipid mixture composed of DPPC and POPC exhibiting a phase transition at 34 degrees C between a crystalline/liquid-crystalline coexistence region and a completely miscible liquid-crystalline phase was used to control the formation of the trimeric peptide bundle. TH1 is phase excluded in crystalline DPPC domains below 34 degrees C, leading to a larger number of trimers. However, when the DPPC domains are dispersed at temperatures above 34 degrees C, the number of trimers is reduced.     

Combined Monte Carlo and molecular dynamics simulation of hydrated lipid-cholesterol lipid bilayers at low cholesterol concentration

We have applied a hybrid equilibration and sampling procedure for the atomic level simulation of a hydrated lipid bilayer to systems consisting of dipalmitoyl phosphatidylcholine (DPPC) and cholesterol, and palmitoyl-oleyl phosphatidylcholine (POPC) at low (approximately 6%) cholesterol concentration. The procedure is applied to bilayers of 94 molecules of DPPC, 6 molecules of cholesterol, and 3205 water molecules, and to bilayers of 120 molecules of POPC, 8 molecules of cholesterol, and 4268 water molecules, at a temperature of 325 K. After equilibration, three separate 400-ps continuous molecular dynamics runs, separated by 10,000 configurational bias Monte Carlo steps, were carried out for each system. Properties of the systems were calculated and averaged over the three separate runs. Results of the simulations are presented and compared with experimental data and with other recent simulations of DPPC and cholesterol, and of pure DPPC, and pure POPC. Certain properties of the bilayers are indistinguishable from cholesterol-free bilayers, including lateral diffusion and electron density. Other properties, most notably the order parameter profile, show the effect of cholesterol even at low concentrations.     

Time-resolved fluorescence and fourier transform infrared spectroscopic investigations of lateral packing defects and superlattice domains in compositionally uniform cholesterol/phosphatidylcholine bilayers

Time-resolved fluorescence and Fourier transform infrared spectroscopies were used to investigate the lateral organization of lipids in compositionally uniform and fully equilibrated 1-palmitoyl-2-oleoyl-phosphatidylcholine/cholesterol (POPC/CHOL) liposomes prepared by a recently devised low-temperature trapping method. Independent fluorescence decay lifetime and rotational dynamics parameters of diphenylhexatriene (DPH) chain-labeled phosphatidylcholine (DPH-PC) in these liposomes were recovered from the time-resolved fluorescence measurements as a function of cholesterol molar fraction (X(CHOL)) at 23 degrees C. The results indicate significantly greater lifetime heterogeneity, shorter average lifetime, rotational correlation time, and lower order parameter of the DPH moiety at X(CHOL) approximately 0.40 and 0.50 as compared to the adjacent cholesterol concentrations. Less prominent changes were also detected at, for example, X(CHOL) approximately 0.20 and 0.33. These X(CHOL)'s coincide with the "critical" X(CHOL)'s predicted by the previously proposed superlattice (SL) model, thus indicating that POPC and cholesterol molecules tend to form SL domains where the components tend to be regularly distributed. The data also support another prediction of the SL model, namely that lateral packing defects coexist with the ordered SL domains. It appears that unfavorable interaction of the DPH-moiety of DPH-PC with cholesterol results in a preferential partition of DPH-PC to the defect regions. Fourier transform infrared analysis of the native lipid O=P=O, C=O, and C-H vibrational bands of POPC/CHOL liposomes in the absence of DPH-PC revealed an increase in the conformational order of the acyl chains and a decrease in the conformational order (or increased hydration) of the interfacial and headgroup regions at or close to the predicted critical X(CHOL)'s. This provides additional but probe-independent evidence for SL domain formations in the POPC/CHOL bilayers. We propose that the defect regions surrounding the putative SL domains could play an important role in modulating the activity of various membrane-associated enzymes, e.g., those regulating the lipid compositions of cell membranes.     

Interactions of N-stearoyl sphingomyelin with cholesterol and dipalmitoylphosphatidylcholine in bilayer membranes.

Differential scanning calorimetry and x-ray diffraction have been utilized to investigate the interaction of N-stearoylsphingomyelin (C18:0-SM) with cholesterol and dipalmitoylphosphatidylcholine (DPPC). Fully hydrated C18:0-SM forms bilayers that undergo a chain-melting (gel -->liquid-crystalline) transition at 45 degrees C, delta H = 6.7 kcal/mol. Addition of cholesterol results in a progressive decrease in the enthalpy of the transition at 45 degrees C and the appearance of a broad transition centered at 46.3 degrees C; this latter transition progressively broadens and is not detectable at cholesterol contents of >40 mol%. X-ray diffraction and electron density profiles indicate that bilayers of C18:0-SM/cholesterol (50 mol%) are essentially identical at 22 degrees C and 58 degrees C in terms of bilayer periodicity (d = 63-64 A), bilayer thickness (d rho-p = 46-47 A), and lateral molecular packing (wide-angle reflection, 1/4.8 A-(1)). These data show that cholesterol inserts into C18:0-SM bilayers, progressively removing the chain-melting transition and altering the bilayer structural characteristics. In contrast, DPPC has relatively minor effects on the structure and thermotropic properties of C18:0-SM. DPPC and C18:0-SM exhibit complete miscibility in both the gel and liquid-crystalline bilayer phases, but the pre-transition exhibited by DPPC is eliminated at >30 mol% C18:0-SM. The bilayer periodicity in both the gel and liquid-crystalline phases decreases significantly at high DPPC contents, probably reflecting differences in hydration and/or chain tilt (gel phase) of C18:0-SM and DPPC.     

Optimization of receptor-G protein coupling by bilayer lipid composition I: kinetics of rhodopsin-transducin binding.

The role of membrane composition in modulating the rate of G protein-receptor complex formation was examined using rhodopsin and transducin (G(t)) as a model system. Metarhodopsin II (MII) and MII-G(t) complex formation rates were measured, in the absence of GTP, via flash photolysis for rhodopsin reconstituted in 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (18:0,18:1PC) and 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0,22:6PC) bilayers, with and without 30 mol% cholesterol. Variation in bilayer lipid composition altered the lifetime of MII-G(t) formation to a greater extent than the lifetime of MII. MII-G(t) formation was fastest in 18:0,22:6PC and slowest in 18:0,18:1PC/30 mol% cholesterol. At 37 degrees C and a G(t) to photolyzed rhodopsin ratio of 1:1 in 18:0,22:6PC bilayers, MII-G(t) formed with a lifetime of 0.6 +/- 0.06 ms, which was not significantly different from the lifetime for MII formation. Incorporation of 30 mol% cholesterol slowed the rate of MII-G(t) complex formation by about 400% in 18:0,18:1PC, but by less than 25% in 18:0,22:6PC bilayers. In 18:0,22:6PC, with or without cholesterol, MII-G(t) formed rapidly after MII formed. In contrast, cholesterol in 18:0,18:1PC induced a considerable lag time in MII-G(t) formation after MII formed. These results demonstrate that membrane composition is a critical factor in determining the temporal response of a G protein-coupled signaling system.     

The effect of cholesterol in a liposomal Muc1 vaccine

A liposomal Muc1 mucin vaccine for treatment of adenocarcinomas was formulated by incorporating a synthetic Muc1 mucin-based lipopeptide and Lipid A into a DPPC/cholesterol bilayer. Vaccination of mice with the liposomal formulation produced a peptide-specific immune response dependent on the cholesterol content. The response occurred at a threshold of 20-23 mol% cholesterol, and was optimal at cholesterol levels of > or =30 mol%. To understand this cholesterol dependency, we studied the effect of cholesterol on the liposomal bilayer and surface properties. Freeze-fracture electron microscopy showed a unique surface texture that was codependent upon cholesterol (> or =20 mol%) and lipopeptide content. Fluorescence anisotropy measurements exhibited a significant decrease in the rotational motion of 1,6-diphenyl-1,3,5-hexatriene in formulations containing >20 mol% cholesterol and only in the presence of the lipopeptide. At 20 mol% cholesterol and with lipopeptide, DSC showed a significant increase in the main phase transition of the DPPC bilayers, while Raman spectroscopy indicated a more ordered arrangement of DPPC molecules compared to control liposomes containing DPPC/cholesterol alone. Taken together, the data suggest the presence of lipopeptide-rich microdomains at and above a threshold of 20 mol% cholesterol that may play a role in the induction of a peptide-specific immunological response.     

Location and dynamics of anthracyclines bound to unilamellar phosphatidylcholine vesicles

We have exploited the intrinsic fluorescence properties of the anthracycline antitumor antibiotics to study the dependence on drug structure of relative drug location and dynamics when the anthracyclines were bound to sonicated dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) vesicles at 27.5 degrees C. Iodide quenching experiments at constant ionic strength were used to evaluate the relative accessibilities of the bound fluorophores to membrane-impermeable iodide. Iodide was found to quench the fluorescence of anthracyclines in free solution by both static and dynamic mechanisms, whereas quenching of membrane-bound fluorophores was predominantly due to the dynamic mechanism. Modified Stern-Volmer plots of anthracyclines bound to fluid-phase DMPC bilayers were linear, and the biomolecular rate constant (kq) values ranged from 0.6 X 10(9) to 1.3 X 10(9) M-1 s-1. Modified Stern-Volmer plots of anthracyclines bound to solid-phase DPPC bilayers were curved, indicative of a heterogeneous-bound drug population. A strong correlation between drug hydrophobicity and penetration of the fluorophore into the bilayer was observed for the daunosamine-containing anthracyclines. Steady-state fluorescence anisotropy measurements under iodide quenching conditions were used to investigate the diffusive motions of anthracyclines in isotropic solvent and in fluid-phase DMPC bilayers. Anthracycline derivatives free in solution exhibited limiting anisotropy (alpha infinity) values which decayed to zero at times long compared to the excited-state lifetime, in contrast to anthracyclines bound to fluid-phase DMPC bilayers, which showed nonzero alpha infinity values. Steady-state anisotropies of membrane-bound anthracyclines were found to be governed principally by alpha infinity and not by the mean rotational rate (R).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cationic amphiphiles and the solubilization of cholesterol crystallites in membrane bilayers

Cationic amphiphiles used for transfection can be incorporated into biological membranes. By differential scanning calorimetry (DSC), cholesterol solubilization in phospholipid membranes, in the absence and presence of cationic amphiphiles, was determined. Two different systems were studied: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)+cholesterol (1:3, POPC:Chol, molar ratio) and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (POPS)+cholesterol (3:2, POPS:Chol, molar ratio), which contain cholesterol in crystallite form. For the zwitterionic lipid POPC, cationic amphiphiles were tested, up to 7 mol%, while for anionic POPS bilayers, which possibly incorporate more positive amphiphiles, the fractions used were higher, up to 23 mol%. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and DOTAP in methyl sulfate salt form (DOTAPmss) were found to cause a small decrease on the enthalpy of the cholesterol transition of pure cholesterol aggregates, possibly indicating a slight increase on the cholesterol solubilization in POPC vesicles. With the anionic system POPS:Chol, the cationic amphiphiles dramatically change the cholesterol crystal thermal transition, indicating significant changes in the cholesterol aggregates. For structural studies, phospholipids spin labeled at the 5th or 16th carbon atoms were incorporated. In POPC, at the bilayer core, the cationic amphiphiles significantly increase the bilayer packing, decreasing the membrane polarity, with the cholesterol derivative 3 beta-[N-(N',N'-dimethylaminoethane)-carbamoyl]-cholesterol (DC-chol) displaying a stronger effect. In POPS and POPS:Chol, DC-chol was also found to considerably increase the bilayer packing. Hence, exogenous cationic amphiphiles used to deliver nucleic acids to cells can change the bilayer packing of biological membranes and alter the structure of cholesterol crystals, which are believed to be the precursors to atherosclerotic lesions.     

Combined influence of cholesterol and synthetic amphiphillic peptides upon bilayer thickness in model membranes.

Deuterium (2H) NMR was used to study bilayer hydrophobic thickness and mechanical properties when cholesterol and/or synthetic amphiphillic polypeptides were added to deuterated POPC lipid bilayer membranes in the liquid-crystalline (fluid) phase. Smoothed acyl chain orientational order profiles were used to calculate bilayer hydrophobic thickness. Addition of 30 mol% cholesterol to POPC at 25 degrees C increased the bilayer thickness from 2.58 to 2.99 nm. The peptides were chosen to span the bilayers with more or less mismatch between the hydrophobic peptide length and membrane hydrophobic thickness. The average thickness of the pure lipid bilayers was significantly perturbed upon addition of peptide only in cases of large mismatch, being increased (decreased) when the peptide hydrophobic length was greater (less) than that of the pure bilayer, consistent with the "mattress" model of protein lipid interactions (Mouritsen, O.G., and M. Bloom. 1984. Biophys. J. 46:141-153). The experimental results were also used to examine the combined influence of the polypeptides and cholesterol on the orientational order profile and thickness expansivity of the membranes. A detailed model for the spatial distribution of POPC and cholesterol molecules in the bilayers was proposed to reconcile the general features of these measurements with micromechanical measurements of area expansivity in closely related systems. Experiments to test the model were proposed.     

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.     

Nonpolar interactions between trans-membrane helical EGF peptide and phosphatidylcholines, sphingomyelins and cholesterol. Molecular dynamics simulation studies.

A molecular dynamics simulation study of four lipid bilayers with inserted trans-membrane helical fragment of epithelial growth factor (EGF) receptor (EGF peptide) was performed. The lipid bilayers differ in their lipid composition and consist of (i) unsaturated phosphatidylcholine (palmitoyloleoylphosphatidylcholine, POPC), (ii) POPC and 20 mol% of cholesterol (Chol), (iii) sphingomyelin (SM) and 20 mol% of Chol, and (iv) SM and 50 mol% of Chol. Only 1 out of 26 residues in the EGF-peptide sequence is polar (Thr). The hydrophobic thickness of each bilayer is different but shorter than the length of the peptide and so, due to hydrophobic mismatch, the inserted peptide is tilted in each bilayer. Additionally, in the POPC bilayer, which is the thinnest, the peptide loses its helical structure in a short three-amino acid fragment. This facilitates bending of the peptide and burying all hydrophobic amino acids inside the membrane core (Figure 1(b)). Bilayer lipid composition affects interactions between the peptide and lipids in the membrane core. Chol increases packing of atoms relative to the peptide side chains, and thus increases van der Waals interactions. On average, the packing around the peptide is higher in SM-based bilayers than POPC-based bilayers but for certain amino acids, packing depends on their position relative to the bilayer center. In the bilayer center, packing is higher in POPC-based bilayers, while in regions closer to the interface packing is higher in SM-based bilayers. In general, amino acids with larger side chains interact strongly with lipids, and thus the peptide sequence is important for the pattern of interactions at different membrane depths. This pattern closely resembles the shape of recently published lateral pressure profiles [Ollila et alJ. Struct. Biol. DOI:10.1016/j.jsb.2007.01.012].     

Biophysical perturbations induced by ethylazinphos in lipid membranes

Perturbations induced by ethylazinphos on the physical organization of dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol membranes were studied by differential scanning calorimetry (DSC) and fluorescence polarization of 2-, 6-, 12-(9-anthroyloxy) stearic acids and 16-(9-anthroyloxy) palmitic acid. Ethylazinphos (50 and 100 microM) increases the fluorescence polarization of the probes, either in the gel or in the fluid phase of DPPC bilayers, and this concentration dependent effect decreases from the surface to the bilayer core. Additionally, the insecticide displaces the phase transition to a lower temperature range and broadens the transition profile of DPPC. A shifting and broadening of the phase transition is also observed by DSC. Furthermore at insecticide/lipid molar ratios higher than 1/7, DSC thermograms, in addition to the normal transition centered at 41 degrees C, also display a new phase transition centered at 45.5 degrees C. The enthalpy of this new transition increases with insecticide concentration, with a corresponding decrease of the main transition enthalpy. Ethylazinphos in DPPC bilayers with low cholesterol (< or = 20 mol%) perturbs the membrane organization as described above for pure DPPC. However, cholesterol concentrations higher than 20 mol% prevent insecticide interaction, as revealed by fluorescence polarization and DSC data. Apparently, cholesterol significantly modulates insecticide interaction by competition for similar distribution domains in the membrane. The present results strongly support our previous hypothesis that ethylazinphos locates in the cooperativity region, i.e. the region of C1-C9 atoms of the acyl chains, and extends to the lipid-water interface, where it increases lipid packing order sensed across all the thickness of the bilayer. Additionally, and, on the basis of DSC data, a lateral regionalization of ethylazinphos is here tentatively suggested.