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.     

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.     

Changes in the lipid dynamics of liposomal membranes induced by poly(ethylene glycol): free volume alterations revealed by inter- and intramolecular excimer-forming phospholipid analogs.

Influence of osmotic shrinkage, swelling, and dehydration on large unilamellar liposomes (LUVs) of 1,2-dioleoylsn-glycero-3-phosphocholine (DOPC) was investigated using the fluorescent lipid probes 1-palmitoyl-2-[10-(pyren-1-yl)]-decanoyl-sn-glycero-3-phosphocholi ne (PPDPC) and 1,2-bis[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (bisPDPC). Increasing concentrations of poly(ethylene glycol) (PEG, average molecular weight of 6000) producing osmotic gradients delta omega up to 250 mOsm/kg were first added to the outside of LUV labeled with 0.1 mol% of either of the above fluorescent phospholipids. The resulting osmotic shrinkage was accompanied by a progressive reduction in the lateral diffusion of the membrane-incorporated PPDPC, evident as a decrease in the rate of its intermolecular excimer formation. In contrast, under the same conditions the rate of intramolecular excimer formation by bisPDPC increased. Notably, signals opposite to those described above were observed for both of the fluorescent probes upon osmotic swelling of DOPC liposomes with encapsulated PEG. The lateral diffusion of PPDPC became progressively reduced upon membrane dehydration due to increasing concentrations of symmetrically distributed PEG (with equal polymer concentrations inside and outside of the liposomes) when neither shrinkage nor swelling occurs while enhanced excimer formation by bisPDPC was evident. The later results were interpreted in terms of osmotically induced changes in the hydration of lipids. In brief, the removal of water from the phospholipid hydration shell diminishes the effective size of the polar headgroup, which subsequently allows for an enhanced lateral packing of the phospholipid acyl chains. Our findings are readily compatible with membrane free volume Vf changes due to osmotic forces under three different kinds of stress (shrinkage, swelling, and dehydration) applied on the lipid bilayers.     

Ether phosphatidylcholines: comparison of miscibility with ester phosphatidylcholines and sphingomyelin, vesicle fusion, and association with apolipoprotein A-I

Nonhydrolyzable matrices of ether-linked phosphatidylcholines (PCs) and sphingomyelin have been used to study the mechanism of action of lipolytic enzymes. Since ether PCs, sphingomyelin, and ester PCs vary in the number of hydrogen bond donors and acceptors in the carbonyl region of the bilayer, we have examined several physical properties of ether PCs and sphingomyelin in model systems to validate their suitability as nonhydrolyzable lipid matrices. The intermolecular interactions of ether PCs with ester PCs, sphingomyelin, and cholesterol were investigated by differential scanning calorimetry. Phase diagrams constructed from the temperature dependence of the gel to liquid-crystalline phase transition of 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DPPC-ether) and 1,2-O-ditetradecyl-sn-glycero-3-phosphocholine (DMPC-ether) with both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) demonstrated complete lipid miscibility in the gel and liquid-crystalline phases. Additionally, phase diagrams of egg yolk sphingomyelin (EYSM) with DMPC or DMPC-ether and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-O-dioctadecyl-sn-glycero-3-phosphocholine (DSPC-ether) demonstrated no major differences in miscibility of EYSM in ester and ether PCs. The effect of 10 mol % cholesterol on the thermal transitions of mixtures of ester and ether PCs also indicates little preference of cholesterol for either lipid. The fusion of small single bilayer vesicles of DMPC, DMPC-ether, DPPC, and DPPC-ether to larger aggregates as determined by gel filtration indicated that the ester PC vesicles were somewhat more stable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lowering line tension with high cholesterol content induces a transition from macroscopic to nanoscopic phase domains in model biomembranes

Chemically simplified lipid mixtures are used here as models of the cell plasma membrane exoplasmic leaflet. In such models, phase separation and morphology transitions controlled by line tension in the liquid-disordered (Ld)?+?liquid-ordered (Lo) coexistence regime have been described [1]. Here, we study two four-component lipid mixtures at different cholesterol fractions: brain sphingomyelin (BSM) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol (Chol). On giant unilamellar vesicles (GUVs) display a nanoscopic-to-macroscopic transition of Ld?+?Lo phase domains as POPC is replaced by DOPC, and this transition also depends on the cholesterol fraction. Line tension decreases with increasing cholesterol mole fractions in both lipid mixtures. For the ternary BSM/DOPC/Chol mixture, the published phase diagram [19] requires a modification to show that when cholesterol mole fraction is >~0.33, coexisting phase domains become nanoscopic.     

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.     

Effect of Membrane Composition on Antimicrobial Peptides Aurein 2.2 and 2.3 From Australian Southern Bell Frogs

The effects of hydrophobic thickness and the molar phosphatidylglycerol (PG) content of lipid bilayers on the structure and membrane interaction of three cationic antimicrobial peptides were examined: aurein 2.2, aurein 2.3 (almost identical to aurein 2.2, except for a point mutation at residue 13), and a carboxy C-terminal analog of aurein 2.3. Circular dichroism results indicated that all three peptides adopt an α-helical structure in the presence of a 3:1 molar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPC/DMPG), and 1:1 and 3:1 molar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG). Oriented circular dichroism data for three different lipid compositions showed that all three peptides were surface-adsorbed at low peptide concentrations, but were inserted into the membrane at higher peptide concentrations. The ³¹P solid-state NMR data of the three peptides in the DMPC/DMPG and POPC/POPG bilayers showed that all three peptides significantly perturbed lipid headgroups, in a peptide or lipid composition-dependent manner. Differential scanning calorimetry results demonstrated that both amidated aurein peptides perturbed the overall phase structure of DMPC/DMPG bilayers, but perturbed the POPC/POPG chains less. The nature of the perturbation of DMPC/DMPG bilayers was most likely micellization, and for the POPC/POPG bilayers, distorted toroidal pores or localized membrane aggregate formation. Calcein release assay results showed that aurein peptide-induced membrane leakage was more severe in DMPC/DMPG liposomes than in POPC/POPG liposomes, and that aurein 2.2 induced higher calcein release than aurein 2.3 and aurein 2.3-COOH from 1:1 and 3:1 POPC/POPG liposomes. Finally, DiSC₃5 assay data further delineated aurein 2.2 from the others by showing that it perturbed the lipid membranes of intact S. aureus C622 most efficiently, whereas aurein 2.3 had the same efficiency as gramicidin S, and aurein 2.3-COOH was the least efficient. Taken together, these data show that the membrane interactions of aurein peptides are affected by the hydrophobic thickness of the lipid bilayers and the PG content.     

Effects of lipid chain length and unsaturation on bicelles stability. A phosphorus NMR study

Most studies reported until now on the magnetically alignable system formed by the binary mixtures of long- and short-chain lipids were based on the mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (D14PC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (D6PC) lipids. We have recently shown that a large part of the phase diagrams of this lipid mixture could be understood by taking into account the partial miscibility between the long-chain lipids and the short-chain lipids when the sample was heated above the melting transition temperature (Tm) of the long-chain lipids. In this work, we show by modifying the chain length of either one of the two lipids that it is possible to control their miscibility and thus the intervals of temperature and composition where spontaneous alignment is observed in a magnetic field. By using 31P NMR, we demonstrate that the very special properties of such binary lipid mixtures are correlated with the propensity for short-chain lipids to diffuse into the bilayer regions. We also show that lipid mixtures with comparable properties can be formed with unsaturated lipids such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC).     

Stimulated release of cholesterol from liposomal membranes by a PEGylated phospholipid

PEGylated phospholipids are commonly used to increase the blood-circulation time of liposomes by providing a steric barrier around them. This paper documents a fundamentally new property of these lipids-an ability to stimulate the release of cholesterol from phospholipid membranes. Evidence for such stimulation has been obtained by measuring the transport of dehydroergosterol (DHE), a fluorescent simulant of cholesterol, from donor liposomes made from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-PEG(2000)), and DHE to acceptor liposomes made from POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), and cholesterol. The potential of PEGylated lipids to serve as novel cholesterol-lowering agents is briefly discussed.     

Lipid Organization in Mixed Lipid Membranes Driven by Intrinsic Curvature Difference

Laurdan fluorescence, novel spectral fitting, and dynamic light scattering were combined to determine lateral lipid organization in mixed lipid membranes of the oxidized lipid, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC), and each of the three bilayer lipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). Second harmonic spectra were computed to determine the number of elementary emissions present. All mixtures indicated two emissions. Accordingly, spectra were fit to two log-normal distributions. Changes with PGPC mole fraction, X_PGPC, of the area of the shorter wavelength line and of dynamic light scattering-derived aggregate sizes show that: DPPC and PGPC form component-separated mixed vesicles for X_PGPC ≤ 0.2 and coexisting vesicles and micelles for X_PGPC > 0.2 in gel and liquid-ordered phases and for all X_PGPC in the liquid-disordered phase; POPC and PGPC form randomly mixed vesicles for X_PGPC ≤ 0.2 and component-separated mixed vesicles for X_PGPC > 0.2. DOPC and PGPC separate into vesicles and micelles. Component segregation is due to unstable inhomogeneous membrane curvature stemming from lipid-specific intrinsic curvature differences between mixing molecules. PGPC is inverse cone-shaped because its truncated tail with a terminal polar group points into the interface. It is similar to and mixes with POPC, also an inverse cone because of mobility of its unsaturated tail. PGPC is least similar to DOPC because mobilities of both unsaturated tails confer a cone shape to DOPC, and PGPC separates form DOPC. DPPC and PGPC do not mix in the liquid-disordered phase because mobility of both tails in this phase renders DPPC a cone. DPPC is a cylinder in the gel phase and of moderate similarity to PGPC and mixes moderately with PGPC.     

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.     

A calorimetry and deuterium NMR study of mixed model membranes of 1-palmitoyl-2-oleylphosphatidylcholine and saturated phosphatidylcholines

Binary phase diagrams have been constructed from differential scanning calorimetry (DSC) data for the systems 1-palmitoyl-2-oleylphosphatidylcholine (POPC)/dimyristoylphosphatidylcholine (DMPC), POPC/dipalmitoylphosphatidylcholine (DPPC) and POPC/distearoylphosphatidylcholine (DSPC). Mixtures of POPC with DMPC exhibit complete miscibility in the gel and liquid crystalline states. Mixtures of POPC with DPPC or with DSPC exhibit gel phase immiscibility over the composition range 0-75% DPPC (or DSPC). These results, when taken together with previous studies of mixtures of phosphatidylcholines, are consistent with the hypothesis that PCs whose order-disorder transition temperatures (Tm values) differ by less than 33 deg. C exhibit gel state miscibility. Those whose Tm values differ by more than 33 deg. C exhibit gel state immiscibility. 2H-NMR spectroscopy has been used to further study mixed model membranes composed of POPC and DPPC, in which either lipid has been labeled with deuterium in the 2-, 10- or 16-position of the palmitoyl chain(s) or in the N-methyls of the choline head group. POPC/DPPC mixtures in the liquid crystalline state are intermediate in order between pure POPC and DPPC at the same temperature. The POPC palmitoyl chain is always more disordered than the palmitoyl chains of DPPC in liquid crystalline POPC/DPPC mixtures. This is attributed to the fact that a POPC palmitoyl chain is constrained by direct bonding to have at least one oleyl chain among its nearest neighbors, while a DPPC palmitoyl chain must have at least one neighboring palmitoyl chain. When liquid crystalline POPC, DPPC and POPC/DPPC mixtures are compared at a reduced temperature (relative to the acyl chain order-disorder transition), POPC/DPPC mixtures are more disordered than predicted from the behavior of the pure components, in agreement with enthalpy data derived from DSC studies. Within the temperature range of the broad phase transition of 1:1 POPC/DPPC, a superposition of gel and liquid crystalline spectra is observed for 1:1 POPC/[2H]DPPC, while 1:1[2H]POPC/DPPC exhibits only a liquid crystalline spectrum. Thus, at temperatures within the phase transition region, the liquid crystalline phase is POPC-rich and the gel phase is DPPC-rich. Comparison of the liquid crystalline quadrupole splittings within the thermal phase transition range suggests that mixing of the residual liquid crystalline POPC and DPPC is highly non-ideal.     

Interaction of the antimicrobial peptide pheromone Plantaricin A with model membranes: implications for a novel mechanism of action

Plantaricin A (plA) is a 26-residue bacteria-produced peptide pheromone with membrane-permeabilizing antimicrobial activity. In this study the interaction of plA with membranes is shown to be highly dependent on the membrane lipid composition. PlA bound readily to zwitterionic 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) monolayers and liposomes, yet without significantly penetrating into these membranes. The presence of cholesterol attenuated the intercalation of plA into SOPC monolayers. The association of plA to phosphatidylcholine was, however, sufficient to induce membrane permeabilization, with nanomolar concentrations of the peptide triggering dye leakage from SOPC liposomes. The addition of the negatively charged phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol POPG (SOPC/POPG; molar ratio 8:2) enhanced the membrane penetration of the peptide, as revealed by (i) peptide-induced increment in the surface pressure of lipid monolayers, (ii) increase in diphenylhexatriene (DPH) emission anisotropy measured for bilayers, and (iii) fluorescence characteristics of the two Trps of plA in the presence of liposomes, measured as such as well as in the presence of different quenchers. Despite deeper intercalation of plA into the SOPC/POPG lipid bilayer, much less peptide-induced dye leakage was observed for these liposomes than for the SOPC liposomes. Further changes in the mode of interaction of plA with lipids were evident when also the zwitterionic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphoethanolaminne (POPE) was present (SOPC/POPG/POPE, molar ratio 3:2:5), thus suggesting increase in membrane spontaneous negative curvature to affect the mode of association of this peptide with lipid bilayer. PlA induced more efficient aggregation of the SOPC/POPG and SOPC/POPG/POPE liposomes than of the SOPC liposomes, which could explain the attenuated peptide-induced dye leakage from the former liposomes. At micromolar concentrations, plA killed human leukemic T-cells by both necrosis and apoptosis. Interestingly, plA formed supramolecular protein-lipid amyloid-like fibers upon binding to negatively charged phospholipid-containing membranes, suggesting a possible mechanistic connection between fibril formation and the cytotoxicity of plA.     

Application of pressure perturbation calorimetry to lipid bilayers

Pressure perturbation calorimetry (PPC) is a new method that measures the heat consumed or released by a sample after a sudden pressure jump. The heat change can be used to derive the thermal volume expansion coefficient, alpha(V), as a function of temperature and, in the case of phase transitions, the volume change, DeltaV, occurring at the phase transition. Here we present the first report on the application of PPC to determine these quantities for lipid bilayers. We measure the volume changes of the pretransition and main transition of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the thermal expansivity of the fluid phase of DMPC and of two unsaturated lipids, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphocholine. The high sensitivity of PPC instrumentation gives accurate data for alpha(V) and DeltaV even upon the application of relatively low pressures of approximately 5 bar.     

Effect of hydrostatic pressure on water penetration and rotational dynamics in phospholipid-cholesterol bilayers.

The effect of high hydrostatic pressure on the lipid bilayer hydration, the mean order parameter, and rotational dynamics of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) cholesterol vesicles has been studied by time-resolved fluorescence spectroscopy up to 1500 bar. Whereas the degree of hydration in the lipid headgroup and interfacial region was assessed from fluorescence lifetime data using the probe 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), the corresponding information in the upper acyl chain region was estimated from its effect on the fluorescence lifetime of and 3-(diphenylhexatrienyl)propyl-trimethylammonium (TMAP-DPH). The lifetime data indicate a greater level of interfacial hydration for DPPC bilayers than for POPC bilayers, but there is no marked difference in interchain hydration of the two bilayer systems. The addition of cholesterol at levels from 30 to 50 mol% to DPPC has a greater effect on the increase of hydrophobicity in the interfacial region of the bilayer than the application of hydrostatic pressure of several hundred to 1000 bar. Although the same trend is observed in the corresponding system, POPC/30 mol% cholesterol, the observed effects are markedly less pronounced. Whereas the rotational correlation times of the fluorophores decrease in passing the pressure-induced liquid-crystalline to gel phase transition of DPPC, the wobbling diffusion coefficient remains essentially unchanged. The wobbling diffusion constant of the two fluorophores changes markedly upon incorporation of 30 mol% cholesterol, and increases at higher pressures, also in the case of POPC/30 mol% cholesterol. The observed effects are discussed in terms of changes in the rotational characteristics of the fluorophores and the phase-state of the lipid mixture. The results demonstrate the ability of cholesterol to adjust the structural and dynamic properties of membranes composed of different phospholipid components, and to efficiently regulate the motional freedom and hydrophobicity of membranes, so that they can withstand even drastic changes in environmental conditions, such as high external hydrostatic pressure.     

Macroscopic consequences of the action of phospholipase C on giant unilamellar liposomes

Macroscopic consequences of the formation of diacylglycerol by phospholipase C (PC-PLC) in giant 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) unilamellar vesicles (GUVs, diameter 10-100 microm) were studied by phase contrast and fluorescence microscopy. PC-PLC caused a series of fast stepwise shrinkages of fluid SOPC GUVs, continuing until the vesicle disappeared beyond the optical resolution of the microscope. The presence of N-palmitoyl-sphingomyelin (mole fraction X = 0.25) in the GUVs did not affect the outcome of the PC-PLC reaction. In addition to hydrolysis, PC-PLC induced adhesion of vicinal vesicles. When multilamellar SOPC vesicles were used only a minor decrease in their diameter was evident suggesting that PC-PLC can exert its hydrolytic activity only in the outer monolayer. A series of stepwise shrinkages was observed also for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) GUVs above their main phase transition temperature, T(m), i.e., when the bilayer is in the liquid crystalline state. However, this process was not observed for DMPC GUVs in the gel state, below T(m). These results are supported by the enhanced activity of PC-PLC upon exceeding T(m) of DMPC large unilamellar vesicles (diameter approximately 0.1 microm) used as a substrate. Studies on SOPC monolayers revealed that PC-PLC can exert its hydrolytic activity only at surface pressures below approximately 30 mN/m. Accordingly, the lack of changes in the gel state DMPC GUVs could be explained by the equilibrium lateral pressure in these vesicles exceeding this critical value.     

Effects of lactose permease of Escherichia coli on the anisotropy and electrostatic surface potential of liposomes

The membrane transport protein lactose permease (LacY), a member of the Major Facilitator Superfamily (MFS) containing twelve membrane-spanning segments connected by hydrophilic loops, was reconstituted in liposomes of: (i) 1,2-dimyristoyl-sn-glycero-3-phosphocoline (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in equimolar proportions; and (ii) Escherichia coli total lipid extract. The structural order of the lipid membranes, in the presence and absence of LacY, was investigated using steady-state fluorescence anisotropy. The features of the anisotropy curves obtained with 1,6-phenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluene sulfonate (TMA-DPH) evidenced: (i) the insertion of LacY into the bilayer; and (ii) a surface effect on the membranes. The most dramatic effects were observed when LacY was reconstituted in the E. coli lipid matrix. The effect of the protein on the electrostatic surface potential of each bilayer was also examined using a fluorescent pH indicator, 4-Heptadecyl-7-hydroxycoumarin (HHC). Changes in surface potential were enhanced in the presence of the substrate (i.e. lactose) only when the lipid matrices were charged. These results suggest a role for charged phospholipids (i.e. phosphatidylethanolamine or phosphatidylglycerol) in proton transfer to the amino acids involved in substrate translocation.     

Location of the TEMPO moiety of TEMPO-PC in phosphatidylcholine bilayers is membrane phase dependent

The (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) moiety tethered to the headgroup of phosphatidylcholine (PC) lipid is employed in spin labeling electron paramagnetic resonance spectroscopy to probe the water dynamics near lipid bilayer interfaces. Due to its amphiphilic character, however, TEMPO spin label could partition between aqueous and lipid phases, and may even be stabilized in the lipid phase. Accurate assessment of the TEMPO-PC configuration in bilayer membranes is essential for correctly interpreting the data from measurements. Here, we carry out all-atom molecular dynamics (MD) simulations of TEMPO-PC probe in single-component lipid bilayers at varying temperatures, using two standard MD force fields. We find that, for a dipalmitoylphosphatidylcholine (DPPC) membrane whose gel-to-fluid lipid phase transition occurs at 314 K, while the TEMPO spin label is stabilized above the bilayer interface in the gel phase, there is a preferential location of TEMPO below the membrane interface in the fluid phase. For bilayers made of unsaturated lipids, 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which adopt the fluid phase at ambient temperature, TEMPO is unequivocally stabilized inside the bilayers. Our finding of membrane phase-dependent positioning of the TEMPO moiety highlights the importance of assessing the packing order and fluidity of lipids under a given measurement condition.     

Optimizing oriented planar-supported lipid samples for solid-state protein NMR

Sample orientation relative to the static magnetic field of an NMR spectrometer allows study of membrane proteins in the lipid bilayer setting. The straightforward preparation and handling of extremely thin mica substrates with consistent surface properties has prompted us to examine oriented phospholipid bilayer and hexagonal phases on mica. The spectral characteristics of oriented lipid samples formed on mica are as good as or better than those on glass. Nine solvents with varying dielectric constants were used to cast lipid films or for vesicle spreading; film characteristics were then compared, and static solid-state 31P-NMR was used to characterize the degree of orientation of the hydrated lipid species. Lipids with four headgroup chemistries were tested: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Solvent affected orientation of POPG, DOPA, and DOPE, but not POPC. Film characteristics varied with solvent, with ramifications for producing homogeneous oriented lipid samples. POPC was used to optimize the amount of lipid per substrate and compare hydration methods. POPG did not orient reproducibly, whereas POPG-POPC mixtures did. DOPA showed 1-2 oriented states depending upon hydration level and deposition method. DOPE formed an oriented hexagonal phase that underwent a reversible temperature-induced phase transition to the oriented bilayer phase.