Osmometric and microscopic studies on bilayers of polar lipids from the extreme halophile, halobacterium cutirubrum

The major membrane polar lipid components in Halobacterium cutirubrum are the diphytanyl ether analogues of phosphatidylglycerol phosphate, glycolipid sulfate, phosphatidylglycerol and phosphatidylglycerol sulfate. Dispersions of total polar lipids in water formed large birefringent liposomes showing concentric lipid bilayers in the elctron microscope; they behaved as ideal osmometers in KCl or NaCl solutions in the concentration range 0.005–0.2 M. At concentrations above 0.2 M KCl the liposomes shrank to spherical particles which were much less birefringent, showed no distinct bilayer structures by electron microscopy, and no longer behaved as ideal osmometers. Dispersions of phosphatidylglycerol phosphate, phosphatidylglycerol or phosphatidylglycerol sulfate alone did not behave as osmometers at any concentration of KCl or NaCl, but glycolipid sulfate alone or mixed with phosphatidylglycerol phosphate or phosphatidylglycerol phosphate + phosphatidylglycerol sulfate showed ideal osmometer behavior in 0.005–0.2 M KCl or NaCl. The highly negatively charged total polar lipids of H. cutirubrum thus can form stable lipid bilayers only at low ionic concentrations (0.005–0.2 M), much lower than the salt concentration (4 M) of the growth medium, and the presence of glycolipid sulfate is essential. Stability of the membrane in 4 M salt appears to require direct participation of the protein components.     

Phase separations induced by melittin in negatively-charged phospholipid bilayers as detected by fluorescence polarization and differential scanning calorimetry

Interactions between melittin and a variety of negatively-charged lipid bilayers have been investigated by intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene and differential scanning calorimetry. (1) Intrinsic fluorescence of the single tryptophan residue of melittin shows that binding of this peptide to negatively-charged phospholipids is directly related to the surface charge density, but is unaffected by the physical state of lipids, fluid or gel, single-shell vesicles or unsonicated dispersions. (2) Changes in the thermotropic properties of negatively-charged lipids upon melittin binding allow to differentiate two groups of lipids: (i) A progressive disappearance of the transition, without any shift in temperature, is observed with monoacid C₁₄ lipids such as dimyristoylphosphatidylglycerol and -serine (group 1). (ii) With a second group of lipids (group 2), a transition occurs even at melittin saturation, and two transitions are detected at intermediate melittin content, one corresponding to remaining unperturbed lipids, the other shifted downward by 10–20°C. This second group of lipids is constituted by monoacid C₁₆ lipids, dipalmitoylphosphatidylglycerol and -serine. Phosphatidic acids also enter this classification, but it is the net charge of the phosphate group which allows to discriminate: singly charged phosphatidic acids belong to group 2, whereas totally ionized ones behave like group 1 lipids, whatever the chain length. (3) It is concluded that melittin induces phase separations between unperturbed lipid regions which give a transition at the same temperature as pure lipid, and peptide rich domains in which the stoichiometry is 1 toxin per 8 phospholipids. The properties of such domains depend on the bilayer stability: in the case of C₁₆ aliphatic chains and singly charged polar heads, the lipid-peptide domains have a transition at a lower temperature than the pure lipid. With shorter C₁₄ chains or with two net charges by polar group, the bilayer structure is probably totally disrupted, and the new resulting phase can no longer lead to a cooperative transition.     

The activation energy of incorporation of extrinsic probes in model vesicles

Time dependence of fluorescence enhancement of probes after addition to lipid vesicles has been used to investigate the position of chromophores in the lipid bilayer. Incorporation studies of a series of $\text{n-(9-}\text{anthroyloxy}$) fatty acids ($\text{n}\text{= 2, 2, 12}\text{and}\text{16}$) and 1,6-diphenylhexatriene in dipalmitoyl phosphatidylcholine vesicles are described. The activation energies for incorporation of these several lipid-mimic type fluorescent probes have been measured. Results show that the activation energy is a function of the distance of the anthracene moiety (chromophore) from the polar end of the probe and the length of the acyl portion of the probe. An average insertion energy of 0.6 kcal/carbon is seen for these fatty acid probes. The activation energy of 1,6-diphenylhexatriene, a factor of 2 greater than that of 16-(9-anthroyloxy)palmitic acid, is consistent with locating 1,6-diphenyl-hexatriene in the middle of the bilayer.     

The structure of membrane lipids of the extreme halophile,Halobacterium cutirubrum,in aqueous systems studied by freeze-fracture

The structures formed by the two major membrane lipids of the extreme halophile, Halobacterium cutirubrum, namely diphytanyl ether analogues of phosphatidylglycerol phosphate and glycolipid sulphate, dispersed in either water, 1 M NaCl or 5 M NaCl were examined by freeze-fracture electron microscopy. In water, both lipids formed lamellar phases which were highly hydrated. Dispersion in 1 M NaCl caused the bilayers to stack more tightly. The presence of 5 M NaCl, mixed phases were observed at 20°C consisting of both lamellar and non-lamellar structures. Studies of binary mixtures of the two lipids in 5 M NaCl in mole ratios of 1:2, 2:1 and 3.5:1 indicated that phase separation takes place and that glycolipid sulphate tended to form bilayers at the growth temperature whereas phosphatidylglycerol phosphate preferentially formed a non-bilayer arrangement in the presence of salt. Total polar lipid extracts H. cutirubrum formed mixed phase systems that reflected the proportions of the major lipid components. Thermotropic studies performed by thermally quenching dispersions at temperatures ranging from −30°C to 70°C indicated that bilayers were formed at lower temperatures in both pure lipids and mixtures of lipids whereas there was a preference for what gave the appearance of inverted cubic phases at high temperatures. These observations are consistent with the notion that non-bilayer lipids are required to package the intrinsic membrane proteins into a lipid bilayer matrix.     

Critical point for membrane bilayer formation

Unilamellar liposomes often are employed in investigations of lipid-protein interactions and the delivery of drugs in therapies for disease. Also, related lipid-containing nanoparticles have been developed as elements of a new class of mRNA vaccines. We show that only unilamellar films form in equilibrium lipid dispersions, at temperature values {T*} that depend on the identities of the lipids (e.g., T* ≈ 29 °C for DMPC). Thermodynamic analysis confirms that films at air-water surfaces can be used to monitor the properties of the lipid vesicles that form in the dispersion. When T > T*, critical exponents describing film properties as T approaches T* are μ ≈ 1.4 and ν ≈ 0.7, which are close to values for the interfacial tension and the correlation length of density fluctuations at fluid interfaces. These results, and observations that within the bilayer the lateral diffusion of fluorescent lipid probes demonstrates increases at T*, suggest that unilamellar vesicles at T* are a transition state between two different multilamellar structures. We generalize the thermodynamic arguments to explain the linkage between lipid structures in the surface and bulk dispersion within more complex samples, showing that dispersions containing total lipid extracts of cell membranes have properties similar to those in dispersions containing single lipids. Information from various independent studies indicates that T* noted for bilayer membranes of a population of cells is identical to the temperature at which the growth or gestation of the cells occurs in vivo. Examples include whole-cell lipid extracts obtained from bacteria, and poikilothermic and homeothermic animals.     

Phospholipid phase transitions in homogeneous nanometer scale bilayer discs

Nanoscale protein supported phospholipid bilayer discs, or Nanodiscs, were produced for the purpose of studying the phase transition behavior of the incorporated lipids. Nanodiscs and vesicles were prepared with two phospholipids, dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine, and the phase transition of each was analyzed using laurdan fluorescence and differential scanning calorimetry. Laurdan is a fluorescent probe sensitive to the increase of hydration in the lipid bilayer that accompanies the gel to liquid crystalline phase transition. The emission intensity profile can be used to derive the generalized polarization, a measure of the relative amount of each phase present. Differential scanning calorimetry was used to further quantitate the phase transition of the phospholipids. Both methods revealed broader transitions for the lipids in Nanodiscs compared to those in vesicles. Also, the transition midpoint was shifted 3-4 degrees C higher for both lipids when incorporated into Nanodiscs. These findings are explained by a loss of cooperativity in the lipids of Nanodiscs which is attributable to the small size of the Nanodiscs as well as the interaction of boundary lipids with the protein encircling the discs. The broad transition of the Nanodisc lipid bilayer better mimics the phase behavior of cellular membranes than vesicles, making Nanodiscs a 'native-like' lipid environment in which to study membrane associated proteins.     

Growth inhibition of Halobacterium cutirubrum by cerulenin,a potent inhibitor of fatty acid synthesis

The polar lipids of $\text{Halobacterium cutirubrum}$ are known to consist exclusively of diether derivatives of glycerol, and do not contain fatty acids. However, cerulenin, a specific and potent inhibitor of fatty acid synthesis, was shown to inhibit the growth of this organism. Protection from growth inhibition was demonstrated when fatty acids of 18 carbons were added to the growth medium, but not when palmitic or palmitoleic acids were used. Cerulenin appears to affect synthesis of all polar lipids in this organism while relative levels of protein and nucleic acids were not significantly affected. Growth inhibition by cerulenin supports the conclusion that the fatty acid synthetase system present in $\text{H. cutirubrum}$ is necessary for lipid biosynthesis, despite the fact that fatty acids are not structural components of the lipids of this bacterium. A pathway is proposed to account for these observations.     

Organization and dynamics of NBD-labeled lipids in lipid bilayer analyzed by FRET using the small membrane fluorescent probe AHBA as donor

Fluorescent probes are employed to investigate natural and model membranes. It is important to know probe location and extent of perturbations they cause into the lipid bilayer. Förster Resonance Energy Transfer (FRET) is a useful tool to investigate phenomena involving plasma membranes, and reports in literature used relatively large fluorophores like 1,6-diphenylhexatriene, located at the center of the hydrophobic region, 4-aminophthalimide-based molecules located at lipid/water interfaces and BODIPY-labeled phosphatidylcholine. In this work we explored FRET process in 1,2-dimyristoyl-L-α-GPC large unilamellar vesicles, in gel and fluid phase, using as donor the very small group o-Abz bound to hexadecyl chain (2-amino-N-hexadecyl-benzamide - AHBA) and 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) labeled lipids as acceptor. From the intensity decay of donor in presence of acceptors, the FRET efficiency was calculated, and used to fit the model proposed by Fung and Stryer to that efficiency. Using lipid bilayer structural data, the procedure allowed the determination of Förster distance for each donor-acceptor pair in vesicles, without imposing any value for the orientational factor κ². From distance distributions between o-Abz in AHBA and NBD in lipid bilayer obtained using the program CONTIN, we obtained donor-acceptor populations having different separation distances. The populations reflect the occurrence of FRET involving probes in the same or in opposite leaflet. A dynamic picture emerged showing how relative position of the probes is dependent on the structural thermal phase of the DMPC bilayer. The results emphasize the need of careful analysis in order to understand processes involving fluorescent probes in model membranes.     

Chemistry and biology of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled lipids: fluorescent probes of biological and model membranes

Lipids that are covalently labeled with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group are widely used as fluorescent analogues of native lipids in model and biological membranes to study a variety of processes. The fluorescent NBD group may be attached either to the polar or the apolar regions of a wide variety of lipid molecules. Synthetic routes for preparing the lipids, and spectroscopic and ionization properties of these probes are reviewed in this report. The orientation of various NBD-labeled lipids in membranes, as indicated by the location of the NBD group, is also discussed. The NBD group is uncharged at neutral pH in membranes, but loops up to the surface if attached to acyl chains of phospholipids. These lipids find applications in a variety of membrane-related studies which include membrane fusion, lipid motion and dynamics, organization of lipids and proteins in membranes, intracellular lipid transfer, and bilayer to hexagonal phase transition in liposomes. Use of NBD-labeled lipids as analogues of natural lipids is critically evaluated.     

Organization and dynamics of lipids in bovine brain coated and uncoated vesicles

Three characteristics have been demonstrated by the chemical analysis of bovine brain coated vesicles following removal of the coat proteins: a high protein content, a high cholesterol/lipid ratio and a high percentage of phosphatidylethanolamine amongst the phospholipids.The study of lipid bilayer organization and dynamics has been performed using the fluorescent probes pyrene and parinaric acid (cis and trans). This has allowed the study of both lateral mobility and rotational motion in the lipid bilayer of the coated and uncoated vesicles.Lateral mobility in the fluid phase of the lipid is slightly reduced by the presence of the clathrin coat, as indicated by the lower diffusion coefficient of pyrene in coated compared with uncoated vesicles.At all temperatures from 6° to 30°C, solid-phase domains, probed by trans parinaric acid, coexist with fluid-phase domains in the lipid bilayer. The temperature dependence of the parinaric acid lifetimes and of their amplitudes strongly suggests that the solid phase domains decrease in size with temperature, both in coated and uncoated vesicles.However, the difference in the value of the anisotropy at long times (r ), between coated and uncoated vesicles (a difference which is more pronounced for cis than for trans parinaric acid), indicates that the presence of the clathrin coat introduces disorder in the surrounding lipids, thus suggesting a possible role of the clathrin in the formation of the pits on the plasma membrane.Abbreviations CVs coated vesicles - UVs uncoated vesicles - TLC thin layer chromatography - DMSO dimethylsulfoxide - DPPC dipalmitoylphosphatidylcholine - cis Pna cis parinaric acid - (9,11,13,15-cis-trans-trans-cis) octadecatetraenoic acid - Trans Pna Trans parinaric acid - (9,11,13,15-all-trans) octadecatetraenoic acid     

Effect of the vesicular stomatitis virus matrix protein on the lateral organization of lipid bilayers containing phosphatidylglycerol: use of fluorescent phospholipid analogues

In order to investigate the mode of interaction of peripheral membrane proteins with the lipid bilayer, the basic (pI approximately 9.1) matrix (M) protein of vesicular stomatitis virus was reconstituted with small unilamellar vesicles (SUV) containing phospholipids with acidic head groups. The lateral organization of lipids in such reconstituted membranes was probed by fluorescent phospholipid analogues labeled with pyrene fatty acids. The excimer/monomer (E/M) fluorescence intensity ratios of the intrinsic pyrene phospholipid probes were measured at various temperatures in M protein reconstituted SUV composed of 50 mol % each of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). The M protein showed relatively small effects on the E/M ratio either in the gel or in the liquid-crystalline phase. However, during the gel to liquid-crystalline phase transition, the M protein induced a large increase in the E/M ratio due to phase separation of lipids into a neutral DPPC-rich phase and DPPG domains presumably bound to M protein. Similar phase separation of bilayer lipids was also observed in the M protein reconstituted with mixed lipid vesicles containing one low-melting lipid component (1-palmitoyl-2-oleoylphosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylglycerol) or a low mole percent of cholesterol. The self-quenching of 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence, as a measure of lipid clustering in the bilayer, was also studied in M protein reconstituted DPPC-DPPG vesicles containing 5 mol % NBD-phosphatidylethanolamine (NBD-PE). The quenching of NBD-PE was enhanced at least 2-fold in M protein reconstituted vesicles at temperatures within or below the phase transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reaction characteristics and mechanisms of lipid bilayer vesicle fusion

Small unilamellar lipid bilayer vesicles were prepared from brain phosphatidylserine, egg phosphatidylcholine, and synthetic dipalmitoylphosphatidylcholine, and were fused into larger structures by freezing and thawing, addition of calcium chloride, and passage through the lipid phase transition temperature. Fusion reactions were studied by electron microscopy, light scattering, and use of fluorescent probes. Fusion was accompanied by leakage of lipid vesicle constituents and of water-soluble solutes in the inner vesicle compartments, and by uptake of these types of components from the external solution. Such leakage was greater during fusion by freezing than by Ca²⁺. Passage through the transition temperature produced a moderate degree of fusion, without loss of membrane components. It is concluded that each fusion method gives rise to a characteristic size or narrow range of sizes of fusion products. The fraction of small vesicles fused into larger structure depends on the method of vesicle preparation, composition of the lipid bilayer, and composition of the external solution. Fusion is induced by creation of a discontinuity in the bilayer or by removal of water associated with the bilayer. The amount of water removed controls the extent of fusion. This is maximized in bilayers when in the liquid-crystal phase, as against the gel phase, in vesicles made by ethanol injection, as against sonication, and in charged bilayers, as against neutral ones.     

Interactions of pyrethroids with phosphatidylcholine liposomal membranes

Interactions of several pyrethroids with membrane lipids in the form of dipalmitoylphosphatidylcholine (DPPC) liposomes have been studied using fluorescent membrane probes. Fluorescence anisotropy values and lifetimes (determined by phase-shift and demodulation techniques) of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene, were decreased in gel phase liposomes by pyrethroids at concentrations on the order of 10 microM. The pyrethroids containing a cyano substituent were also observed to cause collisional quenching of diphenylhexatriene fluorescence. Pyrethroids differed in their effectiveness at lowering the phase transition temperature of DPPC, and in their ability to broaden the temperature range of this transition. The fluorescence intensity of DPPC-incorporated chlorophyll a was used to monitor the pretransition of DPPC and the lateral diffusion of a membrane component located in the polar headgroup region. Permethrin did not affect chlorophyll a fluorescence intensity at any temperature. It may be concluded from these results that pyrethroids are preferentially located in the interior hydrophobic regions of the lipid bilayer, and that these compounds can disorder hydrocarbon packing in the bilayer core. However, polar headgroups were not disordered, and diffusion of membrane components in the polar headgroup region was not altered.     

A model for the packing of lipids in bilayer membranes

A number of known structural properties of mixed lipid bilayer membranes and monolayers are accounted for by a model in which lipids pack into bilayers and monolayers like building blocks, each characterized by a surface head group area and characteristic solid angle. In phospholipids above the melting transition the head group area (at a given temperature and degree of hydration) is fairly invariant while the hydrocarbon region may be liquid-like so long as the molecule is not compressed beyond its characteristic solid angle.Phosphotidylcholine and phosphotidylserine are tapered lipids, i.e. their surface head group areas are greater than their non-polar end areas; cholesterol is frayed, i.e. its polar end area is less than its non-polar end area; while phosphotidylethanolamine is almost cylindrical. The “condensing” effect of cholesterol in mixed phospholipid-cholesterol films is seen as a taper-fray accomodation. The lipid distribution in erythrocyte membranes is shown to be conductive to a stable strain-free membrane.     

Homogeneous catalytic hydrogenation of the polar lipids of pea chloroplasts in situ and the effects on lipid polymorphism

The pattern of hydrogenation of polar lipids of pea chloroplasts incubated in the presence of the homogeneous catalyst Pd(QS)₂, a sulphonated alizarine complex of Pd(II) has been examined. Analysis of the fatty acyl residues of the major lipid classes from chloroplast suspensions at intervals during incubation under hydrogenating conditions showed that susceptibility to hydrogenation increased in the order monogalactosyldiacylglycerol > digalactosyldiacylglycerol > sulphoquinovosyldiacylglycerol > phosphatidylglycerol. Almost 80% of the total number of double bonds in the polar lipids were removed after 2-h incubation under the conditions employed. The consequence of hydrogenation on the phase behaviour of total polar lipid extracts in aqueous dispersions were examined by freeze-fracture electron microscopy, X-ray diffraction and differential scanning calorimetry. These data indicate that progressive hydrogenation of tne lipids in situ produce a change in the organisation of the lipid when dispersed in water. Single bilayer vesicles are converted to large aggregates of planar bilayer stacks in which the hydrocarbon chains are predominantly in the gel phase configuration. Studies of lipids dispersed in 20 mM MgCl₂ suggest that cohesion between the hydrocarbon chains gradually ameliorates the repulsive effects of the charged lipids, sulphoquinovosyldiacylglycerol and phosphatidylglycerol. This results in the formation of a sheet-like lamellar phase characteristic of dispersions of saturated monogalactosyldiacylglycerols which dominates the total polar lipid extracts of pea chloroplasts.     

A model for the packing of lipids in bilayer membranes.

A number of known structural properties of mixed lipid bilayer membranes and monolayers are accounted for by a model in which lipids pack into bilayers and monolayers like building blocks, each characterized by a surface head group area and characteristic solid angle. In phospholipids above the melting transition the head group area (at a given temperature and degree of hydration) is fairly invariant while the hydrocarbon region may be liquid-like so long as the molecule is not compressed beyond its characteristic solid angle. Phosphatidylcholine and phosphatidylserine are tapered lipids, i.e. their surface head group areas are greater than their non-polar end areas; cholesterol is frayed, i.e. its polar end area is less than its non-polar end area; while phosphatidylethanolamine is almost cylindrical. The "condensing" effect of cholesterol in mixed phospholipid-cholesterol films is seen as a taper-fray accommodation. The lipid distribution in erythrocyte membrane is shown to be conducive to a stable strain-free membrane.     

Fluorescent probes for asymmetric lipid bilayers: Synthesis and properties in phosphatidyl choline liposomes and erythrocyte membranes

Summary We have synthesized three sets of fluorescent probes which we believe will be useful in studies of asymmetric membranes and have studied their interactions with model lipid bilayers and erythrocyte membranes. The probes were designed to partition preferentially into one face of a lipid bilayer with asymmetrically disposed phospholipids and to report lipid transitions in that monolayer.We synthesized more than twenty probes containing anthroyl-, dansyl-, or pyrene rings with acidic, basic, and neutral functional groups and alkyl spacers of various lengths. The interactions of these probes with liposomes of phosphatidyl choline and with erythrocyte membranes were characterized to determine whether probe insertion was asymmetric, how deeply the probe penetrated the bilayer, and whether the probe reflected thermotropic phase transitions in model membranes. The set of variously charged anthroyl esters, analogs of local anaesthetics, appears to be promising for studies of asymmetric membranes.Fluorescent probes have been used extensively to provide information on the lipid regions of biological membranes. Membrane fluidity, a composite of molecular packing and motion of acyl chains in lipid bilayers, has been assessed with a variety of fluorescent probes, the fluorescence of which undergoes some measurable change at the temperature of the membrane's thermotropic phase transition. A large number of fluorescent probes have been used for this purpose. Bashford, Morgan and Radda (Bashford, C.L., Morgan, C.G., Radda, G.K. 1976;Biochim. Biophys. Acta 426: 157) and Thulborn and Sawyer (Thulborn, K.R., Sawyer, W.H. 1978;Biochim. Biophys. Acta 511: 125) synthesized several fatty acid derivatives in which an anthracene group is attached (in ester linkage) along the acyl chain at various positions, and have shown that this set of probes may be useful in probing membrane fluidity at differentdepths within the bilayer.This report describes the synthesis and properties of several sets of amphipathic fluorescent probes, which may partition unequally into the two faces of an asymmetric lipid bilayer, and may therefore provide information about membranes complementary to that obtainable with existing probes.     

Barrier properties of glycophorin-phospholipid systems prepared by different methods

Glycophorin was incorporated into large unilamellar dioleoylphosphatidylcholine vesicles by either a detergent dialysis method using octylglucoside or a method avoiding the use of detergents. The vesicles were characterized and the permeability properties and transbilayer movement of lipids in both vesicles were investigated as a function of the protein concentration and were compared to protein-free vesicles. An insight in the permeability properties of the vesicles was obtained by monitoring the ratio potassium (permeant): dextran (impermeant) trap immediately after separation of the vesicles from the external medium. Glycophorin incorporated without the use of detergents in 1:300 protein:lipid molar ratio induces a high potassium permeability for the majority of the vesicles as judged from the low potassium trap (K⁺:dextran trap = 0.21). In contrast, the vesicles in which glycophorin is incorporated via the octylglucoside method (1:500 protein:lipid molar ratio) are much less permeable to potassium (K⁺:dextran trap = 0.67 and $\text{t}\text{1}\text{2}$ of potassium efflux at 22°C is 7.5 h.). The relationship between protein-induced bilayer permeability and lipid transbilayer movement in both vesicle preparations is discussed. Addition of wheat-germ agglutinin to glycophorin-containing vesicles comprised of dioleoylphosphatidylcholine and total erythrocyte lipids caused no or just a small effect (less than 20% release of potassium) on the potassium permeability of these vesicles. Also, addition of lectin to dioleoylphosphatidylethanolamine-glycophorin bilayer vesicles in a 25:1 lipid:glycophorin molar ratio had no effect on the permeability characteristics of the vesicles. In contrast, addition of wheat-germ agglutinin to bilayer vesicles made of dioleoylphosphatidylethanolamine and glycophorin in a 200:1 molar ratio resulted in a release of 74% of the enclosed potassium by triggering a bilayer to hexagonal (H_II) phase transition. The role of protein aggregation and the formation of defects in the lipid bilayer on membrane permeability and lipid transbilayer movement is discussed.     

Lipid bilayers cushioned with polyelectrolyte-based films on doped silicon surfaces

Silicon semiconductors with a thin surface layer of silica were first modified with polyelectrolytes (polyethyleneimine, polystyrene sulfonate and poly(allylamine)) via a facile layer-by-layer deposition approach. Subsequently, lipid vesicles were added to the preformed polymeric cushion, resulting in the adsorption of intact vesicles or fusion and lipid bilayer formation. To study involved interactions we employed optical reflectometry, electrochemical impedance spectroscopy and fluorescent recovery after photobleaching. Three phospholipids with different charge of polar head groups, i.e. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used to prepare vesicles with varying surface charge. We observed that only lipid vesicles composed from 1:1 (mole:mole) mixture of DOPC/DOPS have the ability to fuse onto an oppositely charged terminal layer of polyelectrolyte giving a lipid bilayer with a resistance of >100 kΩ. With optical reflectometry we found that the vesicle surface charge is directly related to the amount of mass adsorbed onto the surface. An interesting observation was that zwitterionic polar head groups of DOPC allow the adsorption on both positively and negatively charged surfaces. As found with fluorescent recovery after photobleaching, positively charged surface governed by the presence of poly(allylamine) as the terminal layer resulted in intact DOPC lipid vesicles adsorption whereas in the case of a negatively charged silica surface formation of lipid bilayers was observed, as expected from literature.     

Effects of lung surfactant proteolipid SP-C on the organization of model membrane lipids: a fluorescence study.

Lipid-protein interactions of pulmonary surfactant-associated protein SP-C in model DPPC/DPPG and DPPC/DPPG/eggPC vesicles were studied using steady-state and time-resolved fluorescence measurements of two fluorescent phospholipid probes, NBD-PC and NBD-PG. These fluorescent probes were utilized to determine SP-C-induced lipid perturbations near the bilayer surface, and to investigate possible lipid headgroup-specific interactions of SP-C. The presence of SP-C in DPPC/DPPG membrane vesicles resulted in (1) a dramatic increase in steady-state anisotropy of NBD-PC and NBD-PG at gel phase temperatures, (2) a broadening of the gel-fluid phase transition, (3) a decrease in self-quenching of NBD-PC and NBD-PG probes, and (4) a slight increase in steady-state anisotropy of NBD-PG at fluid phase temperatures. Time-resolved measurements, as well as steady-state intensity measurements indicate that incorporation of SP-C into DPPC/DPPG or DPPC/DPPG/eggPC vesicles results in a increase in the fraction of the long-lifetime species of NBD-PC. The results presented here indicate that SP-C orders the membrane bilayer surface, disrupts acyl chain packing, and may increase the lateral pressure within the bilayer.