Comparison of the membrane association of two antimicrobial peptides, magainin 2 and indolicidin

Interactions of two antimicrobial peptides, magainin 2 and indolicidin, with three different model biomembranes, namely, monolayers, large unilamellar vesicles (LUVs), and giant liposomes, were studied. Insertion of both peptides into lipid monolayers was progressively enhanced when the content of an acidic phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) in a film of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) was increased. Indolicidin and magainin 2 penetrated also into lipid monolayers containing cholesterol (mole fraction, X = 0.1). Membrane association of magainin 2 attenuated lipid lateral diffusion in POPG-containing LUVs as revealed by the decrease in the excimer/monomer fluorescence ratio I(e)/I(m) for the pyrene fatty-acid-containing phospholipid derivative 1-palmitoyl-2-[10-(pyren-1-yl) decanoyl]-sn-glycero-3-phospho-rac-glycerol (PPDPG). Likewise, an increase in steady-state fluorescence anisotropy of the membrane-incorporated diphenylhexatriene (DPH) was observed, revealing magainin 2 to increase acyl chain order and induce segregation of acidic phospholipids. Similar effects were observed for indolicidin. The topological effects of magainin 2 and indolicidin on phospholipid membranes were investigated using optical microscopy of giant vesicles. Magainin 2 had essentially no influence on either SOPC or SOPC:cholesterol (X = 0.1) giant liposomes. However, effective vesiculation was observed when acidic phospholipid (X(PG) = 0.1) was included in the giant vesicles. Indolicidin caused only a minor shrinkage of giant SOPC vesicles whereas the formation of endocytotic vesicles was observed when the giant liposome contained POPG (X(PG) = 0.1). Interestingly, for indolicidin, vesiculation was also observed for giant vesicles composed of SOPC/cholesterol (X(chol) = 0.1). Possible mechanisms of membrane transformation induced by these two peptides are discussed.     

Binding of lysozyme to phospholipid bilayers: evidence for protein aggregation upon membrane association

Biological functions of lysozyme, including its antimicrobial, antitumor, and immune-modulatory activities have been suggested to be largely determined by the lipid binding properties of this protein. To gain further insight into these interactions on a molecular level the association of lysozyme to liposomes composed of either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or its mixtures with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-phosphatidylserine, or bovine heart cardiolipin was studied by a combination of fluorescence techniques. The characteristics of the adsorption of lysozyme to lipid bilayers were investigated using fluorescein 5'-isothiocyanate labeled protein, responding to membrane association by a decrease in fluorescence. Upon increasing the content of anionic phospholipids in lipid vesicles, the binding isotherms changed from Langmuir-like to sigmoidal. Using adsorption models based on scaled particle and double-layer theories, this finding was rationalized in terms of self-association of the membrane-bound protein. The extent of quenching of lysozyme tryptophan fluorescence by acrylamide decreased upon membrane binding, revealing a conformational transition for the protein upon its surface association, resulting in a diminished access of the fluorophore to the aqueous phase. Steady-state fluorescence anisotropy of bilayer-incorporated probe 1,6-diphenyl-1,3,5-hexatriene was measured at varying lipid-to-protein molar ratios. Lysozyme was found to increase acyl-chain order for liposomes with the content of acidic phospholipid exceeding 10 mol %. Both electrostatic and hydrophobic protein-lipid interactions can be concluded to modulate the aggregation behavior of lysozyme when bound to lipid bilayers. Modulation of lysozyme aggregation propensity by membrane binding may have important implications for protein fibrillogenesis in vivo. Disruption of membrane integrity by the aggregated protein species is likely to be the mechanism responsible for the cytotoxicity of lysozyme.     

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.     

Atomic force microscopy characterization of supported planar bilayers that mimic the mitochondrial inner membrane

In this study we examined the properties of supported planar bilayers (SPBs) formed from phospholipid components that comprise the mitochondrial inner membrane. We used 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and cardiolipin (CL). Liposomes of binary POPE:POPC (1:1, mol:mol) and ternary (POPE:POPC:CL (0.5:0.3:0.2, mol:mol:mol) composition were used in the formation of SPBs on mica. The characterization of the SPBs was carried out below (4 degrees C) and above (24 and 37 degrees C) the phase transition temperature (Tm) of the mixtures in solution. We observed: (i) that the thickness of the bilayers, calculated from a cross-sectional analysis, decreased as the visualization temperature increased; (ii) the existence of laterally segregated domains that respond to temperature in SPBs of POPE:POPC:CL; (iii) a decrease in height and an increase in roughness (Ra) of SPBs after cytochrome c (cyt c) injection at room temperature. To obtain further insight into the nature of the interaction between cyt c and the bilayers, the competition between 8-anilino-1-naphthalene sulfonate (ANS) and the protein for the same binding sites in liposomes was monitored by fluorescence. The results confirm the existence of preferential interaction of cyt c with CL containing liposomes. Taking these results and those of previous papers published by the group, we discuss the preferential adsorption of cyt c in CL domains. This provides support for the relevance of these phospholipids as a proton trap in the oxidative phosphorylation process that occurs in the energy transducing membranes.     

Poly(ethylene glycol)-induced and temperature-dependent phase separation in fluid binary phospholipid membranes.

Exclusion of the strongly hygroscopic polymer, poly(ethylene glycol) (PEG), from the surface of phosphatidylcholine liposomes results in an osmotic imbalance between the hydration layer of the liposome surface and the bulk polymer solution, thus causing a partial dehydration of the phospholipid polar headgroups. PEG (average molecular weight of 6000 and in concentrations ranging from 5 to 20%, w/w) was added to the outside of large unilamellar liposomes (LUVs). This leads to, in addition to the dehydration of the outer monolayer, an osmotically driven water outflow and shrinkage of liposomes. Under these conditions phase separation of the fluorescent lipid 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) embedded in various phosphatidylcholine matrices was observed, evident as an increase in the excimer-to-monomer fluorescence intensity ratio (IE/IM). Enhanced segregation of the fluorescent lipid was seen upon increasing and equal concentrations of PEG both inside and outside of the LUVs, revealing that osmotic gradient across the membrane is not required, and phase separation results from the dehydration of the lipid. Importantly, phase separation of PPDPC could be induced by PEG also in binary mixtures with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), for which temperature-induced phase segregation of the fluorescent lipid below Tm was otherwise not achieved. In the different lipid matrices the segregation of PPDPC caused by PEG was abolished above characteristic temperatures T0 well above their respective main phase transition temperatures Tm. For 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DMPC, SOPC, and POPC, T0 was observed at approximately 50, 32, 24, and 20 degrees C, respectively. Notably, the observed phase separation of PPDPC cannot be accounted for the 1 degree C increase in Tm for DMPC or for the increase by 0.5 degrees C for DPPC observed in the presence of 20% (w/w) PEG. At a given PEG concentration maximal increase in IE/IM (correlating to the extent of segregation of PPDPC in the different lipid matrices) decreased in the sequence 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) > DPPC > DMPC > SOPC > POPC, whereas no evidence for phase separation in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) LUV was observed (Lehtonen and Kinnunen, 1994, Biophys. J. 66: 1981-1990). Our results indicate that PEG-induced dehydration of liposomal membranes provides the driving force for the segregation of the pyrene lipid.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interaction of the cytoskeletal component vinculin with bilayer structures analyzed with a photoactivatable phospholipid

The cytoskeletal component vinculin has been proposed to act as an actin-plasma membrane linker. In order to demonstrate a possible direct interaction of vinculin with bilayers, photolabeling with a phospholipid generating a highly reactive carbene was used. This phosphatidylcholine analogue (1-palmitoyl-2-[10-[4-[(trifluoromethyl)diazirinyl]phenyl]-[3H] 9-oxaundecanoyl]-sn-glycero-3-phosphocholine), with the photoactivatable diazirine group on its apolar portion, has been shown to label selectively membrane-embedded domains of membrane proteins. Vinculin is significantly labeled upon incubation and photolysis with liposomes containing trace amounts of this photoactivatable phospholipid, but only when the liposomes also contain acidic phospholipids. Labeling of vinculin is markedly increased (5-17-fold) by all acidic phospholipids tested so far (30%, w/w), compared to labeling in neutral phospholipids. Labeling is high at low ionic strength, but significant vinculin labeling can still be observed at physiological salt concentrations and acidic phospholipid content of the membrane. Our results provide evidence that vinculin inserts into the hydrophobic part of the bilayer by interacting with acidic phospholipids. A similar interaction may be of importance in vivo.     

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.     

Phase properties of liquid-crystalline Phosphatidylcholine/Phosphatidylethanolamine bilayers revealed by fluorescent probes.

The mixing properties of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were examined in liquid-crystalline phase using fluorescent probes incorporated into lipid bilayers. The excimer to monomer (E/M) fluorescence ratio of 1-hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (PPC) versus PPC concentration was higher for binary mixtures containing phosphatidylcholine (PC)/phosphatidylethanolamine (PE) (1:1) compared to PC matrix. When POPC was gradually replaced with POPE, the E/M ratio also increased suggesting the enhanced lateral mobility or the lateral enrichment of PPC into domains or both. Evidences for the PE-induced domain formation were further provided by resonance energy transfer between 2-(4, 4-difluoro-5-methyl-4-boro-3a, 4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero- 3-phospho choline and PPC, which was enhanced as a function of PE concentration, and by the polarization of 1,6-diphenyl-1,3, 5-hexatriene. In addition, PE reduced free volume and polarity of lipid bilayers as measured by the emission fluorescence of 1,2-bis PPC and 6-lauroyl-2-dimethylaminonaphthalene. When POPE analogs with a methylated head group instead of normal POPE were used, the diminished effect on the domain formation was shown in the order N-methyl PE > N,N-dimethyl PE. The results suggest that the mixing properties of POPE and POPC are not random but that lipid domains of phospholipids are formed.     

Rapid transbilayer movement of the fluorescent sterol dehydroergosterol in lipid membranes

This study establishes a new assay for measuring the transbilayer movement of dehydroergosterol (DHE) in lipid membranes. The assay is based on the rapid extraction of DHE by methyl-beta-cyclodextrin (M-CD) from liposomes. The concentration of DHE in the liposomal membrane was measured by using fluorescence resonance energy transfer (FRET) from DHE to dansyl-phosphatidylethanolamine, which is not extracted from liposomes by M-CD. The method was applied to small (SUV) and large (LUV) unilamellar vesicles of different compositions and at various temperatures. From the kinetics of FRET changes upon extraction of DHE from membranes, rates of M-CD mediated extraction and flip-flop of DHE could be deduced and were found to be dependent on the physical state of the lipid phase. For egg phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in the liquid-crystalline state, halftimes of extraction and transbilayer movement were <5 s and approximately 20-50 s, respectively, at 10 degrees C. For 1,2-dimyristoyl-sn-glycero-3-phosphocholine-SUV being in the gel state at 10 degrees C, the respective halftimes were 28 s and 5-8 min. Surprisingly, DHE could not be extracted from LUV consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine. This might be an indication of specific interactions between DHE molecules in membranes depending on the phospholipid composition of the membrane.     

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.     

Comparison of the effects of surface tension and osmotic pressure on the interfacial hydration of a fluid phospholipid bilayer

The effects of three so-called kosmotropic solutes, namely, betaine, sucrose, and choline chloride on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine large unilamellar vesicles, were studied by measuring the generalized polarization (GP) for the fluorescence emission of the membrane partitioning probe Laurdan. The latter has been shown to be sensitive to the depth of water penetration into phospholipid bilayers. At equal osmotic pressures the three solutes produced different increments in GP, with a qualitative positive correlation. However, the increments in GP correlated also quantitatively with the increase of air-water surface tension caused by the three kosmotropes. Our findings suggest surface tension to determine the impact of these solutes on the lateral packing of the lipid bilayer. Based on the changes in area/lipid at different surface tensions, the equilibrium lateral pressure for a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer at 25 degrees C was estimated to be approximately 34 mN/m.     

Influence of the physical state of the membrane on the enzymatic activity and energy of activation of protein kinase C alpha.

The activation of protein kinase C alpha was studied by using a lipid system consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) (molar ratio 4:1) and different proportions of 1-palmitoyl-2-oleoyl-sn-glycerol (POG). The phase behavior of the lipidic system was characterized by using differential scanning calorimetry and 31P NMR, and a phase diagram was elaborated. The results suggested the formation of two diacylglycerol/phospholipid complexes, one at 15 mol % of POG and the second at 30 mol % of POG. These two complexes would define the three regions of the phase diagram: in the first region (concentrations of POG lower than 15 mol %) there is gel-gel immiscibility at temperatures below that of the phase transition between C1 and pure phospholipid, and a fluid lamellar phase above of the phase transition. In the second region (between 15 and 30 mol % of POG), gel-gel immiscibility between C1 and C2 with fluid-fluid immiscibility was observed, while inverted hexagonal HII and isotropic phases were detected by 31P NMR. In the third region (concentrations of POG higher than 30 mol %), gel-gel immiscibility seemed to occur between C2 and pure POG along with fluid-fluid immiscibility, while an isotropic phase was detected by 31P NMR. When PKC alpha activity was measured, as a function of POG concentration, maximum activity was found at POG concentrations as low as 5-10 mol %; the activity slightly decreased as POG concentration was increased to 45 mol % at 32 degrees C (above Tc) whereas activity did not change with increasing concentrations of POG at 5 degrees C (below Tc). When the activity was studied as a function of temperature, at different POG concentrations, and depicted as Arrhenius plots, it was found that the activity increased with increasing temperatures, showing a discontinuity at a temperature very close to the phase transition of the system and a lower activation energy at the upper slope of the graph, indicating that the physical state of the membrane affected the interaction of PKC alpha with the membrane.     

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)     

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.     

Interaction of the C2 domain from protein kinase C(epsilon) with model membranes

The C2 domain from protein kinase Cepsilon (PKCepsilon) binds to membranes but does not require Ca2+ to do so. This work examines the mode in which the conformation and organization of the phospholipids present in model membranes are altered by the presence of the C2 domain from PKCepsilon (C2-PKCepsilon). It is concluded from the results of differential scanning calorimetry that the protein shifted the temperature of the gel to the fluid phase transition of pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), widening the transition and increasing it to a higher temperature. When POPA was mixed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), the changes in the transition were smaller and no phase separation was observed. Experiments performed using magic angle spinning NMR showed that this C2 domain specifically affected POPA when the phospholipid was mixed with POPC, as indicated by the downfield shift in the isotropic resonance of POPA, the widening of the resonance peak, the decrease in T2, and the decrease in T1 observed at all temperatures. All these effects were quite marked compared with the very small effect observed with POPC, indicating the specificity of the effect. The presence of the C2-PKCepsilon protein changed the conformation of the polar head group of POPA, as shown by infrared spectroscopy. All these results clearly illustrate the electrostatic interaction that takes place between this C2 domain and membranes which contain POPA in the absence of Ca2+.     

Interaction of the alpha-helices of apolipophorin III with the phospholipid acyl chains in discoidal lipoprotein particles: a fluorescence quenching study.

Quenching of tryptophan fluorescence by nitroxide-labeled phospholipids and nitroxide-labeled fatty acids was used to investigate the lipid-binding domains of apolipophorin III. The location of the Trp residues relative to the lipid bilayer was investigated in discoidal lipoprotein particles made with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and five different single-Trp mutants of apoLp-III. A comparison of the quenching efficiencies of phospholipids containing nitroxide groups at the polar head, and at positions 5 and 16 of the sn-2 acyl chain, indicated that the protein is interacting with the acyl chains of the phospholipid along the periphery of the bilayer of the discoidal lipoprotein. N-Bromosuccinimide readily abolished 100% of the fluorescence of all Trp residues in the lipid-bound state. Larger quenching rates were observed for the Trp residues in helices 1, 4, and 5 than for those located in helices 2 and 3, suggesting differences between the interaction of these two groups of helices. However, the extent of Trp fluorescence quenching observed in lipoproteins made with any of the mutants was comparable to that reported for deeply embedded Trp residues, suggesting that all Trp residues interact with the phospholipid acyl chains. This study provides the first experimental evidence of a massive interaction of the alpha-helices of apoLp-III with the phospholipid acyl chains in discoidal lipoproteins. The extent of interaction deduced is consistent with the apolipoprotein adopting a highly extended conformation.     

Detection of phospholipid phase separation. A multifrequency phase fluorimetry study of 1,6-diphenyl-1,3,5-hexatriene fluorescence

Using multifrequency phase and modulation fluorometry and a nonlinear least-squares analysis of lifetime data, we were able to determine the complex decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in synthetic phospholipid bilayers. Our results showed a monoexponential decay of DPH in the pure isotropic solvents studied, over a wide temperature range, and a double-exponential decay of DPH in phospholipids, both above and below the transition. During the transition, and in mixed-phase phospholipids, a three-component analysis was successfully accomplished, and the pre-exponential factors of the two main components have been shown to be quantitatively representative of the gel and liquid-crystalline phases of the bilayer. The fractional intensity of the shorter lifetime component depends on the modalities of the sample preparation. The factors affecting this component are discussed. From the DPH fluorescence lifetime and from the anisotropy data in L-alpha-dimyristoyl-phosphatidylcholine/L-alpha-dipalmitoyl-phosphatidyl choline mixtures, a phase diagram was independently constructed. Conclusions about the sensitivity and the partition of the probe between gel and the liquid-crystalline phases of the bilayer are derived. Lifetime experiments on DPH in a L-alpha-dilauroyl-phosphatidylcholine/L-alpha-dipalmitoyl-phosphatidylch oline mixture suggested a general method for the determination and quantitation of the two different phases in the bilayer.     

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.     

Contamination of commercial phosphatidylcholine by phospholipase A

During the course of a study involving the assay of a membrane-bound phospholipase A2 it was observed that a commercial preparation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine used as substrate had intrinsic lipolytic activity at pH 8.5. Further investigation revealed a Ca2+-dependent phospholipase A largely susceptible to treatment by the alkylating reagent p-bromophenacyl bromide or by heat (15 min at 120 degrees C). Complete separation of enzyme and phospholipid could be achieved by thin-layer chromatography. Such a contamination was not observed in a chemically identical phosphatidylcholine obtained from a different supplier. These observations may be relevant to investigators using commercial preparations of phospholipids in a variety of studies, including intracellular phospholipase A2 determination.