POPC Vesicles Research Articles

Interaction of phenol-soluble modulins with phosphatidylcholine vesicles.

Several members of the staphylococcal phenol-soluble modulin (PSM) peptide family exhibit pronounced capacities to lyse eukaryotic cells, such as neutrophils, monocytes, and erythrocytes. This is commonly assumed to be due to the amphipathic, α-helical structure of PSMs, giving PSMs detergent-like characteristics and allowing for a relatively non-specific destruction of biological membranes. However, the capacities of PSMs to lyse synthetic phospholipid vesicles have not been investigated. Here, we analyzed lysis of synthetic phosphatidylcholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC) vesicles by all Staphylococcus aureus and S. epidermidis PSMs. In addition, we investigated the lytic capacities of culture filtrates obtained from different S. aureus PSM deletion mutants toward POPC vesicles. Our results show that all staphylococcal PSMs have phospholipid vesicle-lysing activity and the capacity of S. aureus culture filtrate to lyse POPC vesicles is exclusively dependent on PSMs. Notably, we observed largely differing capacities among PSM peptides to lyse POPC vesicles. Interestingly, POPC vesicle-lytic capacities did not correlate with those previously seen for the lysis of eukaryotic cells. For example, the β-type PSMs were strongly lytic for POPC vesicles, but are known to exhibit only very low lytic capacities toward neutrophils and erythrocytes. Thus our results also suggest that the interaction between PSMs and eukaryotic membranes is more specific than previously assumed, potentially depending on additional structural features of those membranes, such as phospholipid composition or yet unidentified docking molecules.

Molecular characterization of the interaction of crotamine-derived nucleolar targeting peptides with lipid membranes

A novel class of cell-penetrating, nucleolar-targeting peptides (NrTPs), was recently developed from the rattlesnake venom toxin crotamine. Based on the intrinsic fluorescence of tyrosine or tryptophan residues, the partition of NrTPs and crotamine to membranes with variable lipid compositions was studied. Partition coefficient values (in the 102–105 range) followed essentially the compositional trend POPC:POPG≤POPG<POPC≤POPC:cholesterol. Leakage assays showed that NrTPs induce minimal lipid vesicle disruption. Fluorescence quenching of NrTPs, either by acrylamide or lipophilic probes, revealed that NrTPs are buried in the lipid bilayer only for negatively-charged membranes. Adoption of partial secondary structure by the NrTPs upon interaction with POPC and POPG vesicles was demonstrated by circular dichroism. Translocation studies were conducted using a novel methodology, based on the confocal microscopy imaging of giant multilamellar vesicles or giant multivesicular liposomes. With this new procedure, which can now be used to evaluate the membrane translocation ability of other molecules, it was demonstrated that NrTPs are able to cross lipid membranes even in the absence of a receptor or transmembrane gradient. Altogether, these results indicate that NrTPs interact with lipid bilayers and can penetrate cells via different entry mechanisms, reinforcing the applicability of this class of peptide as therapeutic tools for the delivery of molecular cargoes.

Formation of Substrate-Supported Membranes from Mixtures of Long- and Short-Chain Phospholipids

We studied the formation of substrate-supported planar phospholipid bilayers (SPBs) on glass and silica from mixtures of long- and short-chain phospholipids to assess the effects of detergent additives on SPB formation. 1,2-Hexyanoyl-sn-glycero-3-phosphocholine (DHPC-C6) and 1,2-heptanoyl-sn-glycero-3-phosphocholine (DHPC-C7) were chosen as short-chain phospholipids. 1-Palmitoyl-2-oleol-sn-glycero-3-phosphocholine (POPC) was used as a model long-chain phospholipid. Kinetic studies by quartz crystal microbalance with dissipation monitoring (QCM-D) showed that the presence of short-chain phospholipids significantly accelerated the formation of SPBs. Rapid rinsing with a buffer solution did not change the adsorbed mass on the surface if POPC/DHPC-C6 mixtures were used below the critical micelle concentration (cmc) of DHPC-C6, indicating that an SPB composed of POPC molecules remained on the surface. Fluorescence microscopy observation showed homogeneous SPBs, and the fluorescence recovery after photobleaching (FRAP) measurements gave a diffusion coefficient comparable to that for SPBs formed from POPC vesicles. However, mixtures of POPC/DHPC-C7 resulted in a smaller mass of lipid adsorption on the substrate. FRAP measurements also yielded significantly smaller diffusion coefficients, suggesting the presence of defects. The different behaviors for DHPC-C6 and DHPC-C7 point to the dual roles of detergents to enhance the formation of SPBs and to destabilize them, depending on their structures and aggregation properties.

Elucidating Driving Forces for Liposome Rupture: External Perturbations and Chemical Affinity

Atomic force microscopy (AFM) studies under aqueous buffer probed the role of chemical affinity between liposomes, consisting of large unilamellar vesicles, and substrate surfaces in driving vesicle rupture and tethered lipid bilayer membrane (tLBM) formation on Au surfaces. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-poly(ethylene glycol)-2000-N-[3-(2-pyridyldithio) propionate] (DSPE-PEG-PDP) was added to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles to promote interactions via Au-thiolate bond formation. Forces induced by an AFM tip leading to vesicle rupture on Au were quantified as a function of DSPE-PEG-PDP composition with and without osmotic pressure. The critical forces needed to initiate rupture of vesicles with 2.5, 5, and 10 mol % DSPE-PEG-PDP are approximately 1.1, 0.8, and 0.5 nN, respectively. The critical force needed for tLBM formation decreases from 1.1 nN (without osmotic pressure) to 0.6 nN (with an osmotic pressure due to 5 mM of CaCl(2)) for vesicles having 2.5 mol % DSPE-PEG-PDP. Forces as high as 5 nN did not lead to LBM formation from pure POPC vesicles on Au. DSPE-PEG-PDP appears to be important to anchor and deform vesicles on Au surfaces. This study demonstrates how functional lipids can be used to tune vesicle-surface interactions and elucidates the role of vesicle-substrate interactions in vesicle rupture.

Thermodynamic Measurements of Bilayer Insertion of a Single Transmembrane Helix

Accurate determination of the free energy of transfer of a helical segment from aqueous into a transmembrane conformation is essential for understanding and predicting of the folding and stability of membrane proteins. Until recently, direct thermodynamically sound measurements of free energy of insertion of hydrophobic transmembrane peptides were impossible due to peptides’ aggregation outside the lipid bilayer. Here we overcome this problem by using fluorinated surfactants that are capable of preventing aggregation, but, unlike detergents, do not themselves interact with the bilayer. We have applied previously introduced FCS (Fluorescence Correlation Spectroscopy) methodology [Posokhov et al., Biophysical J. 2008, 95:L54-56] to study surfactant-chaperoned insertion into preformed POPC vesicles of the two well-studied dye-labeled transmembrane peptides of different lengths: WALP23 and WALP27. Interpolation of the apparent free energy values measured in the presence of surfactants to a zero surfactant concentration yielded free energy values of −9.0 and −10.0 kcal/mole for insertion of WALP23 and WALP27, respectively. Circular dichroism measurements confirmed a predominantly helical structure of peptides in lipid bilayer, in the presence of surfactants and in aqueous mixtures of organic solvents. From a combination of thermodynamic and conformational measurements we conclude that the partitioning of a 4-residue LALA segment in the context of a continuous helical conformation from aqueous environment into the hydrocarbon core of the membrane has a favorable free energy of 1 kcal per mole. Our measurements combined with the predictions of the Wimley-White hydrophobicity scale indicates that the per residue cost of the helical backbone partitioning is unfavorable and equals +0.13 kcal/mole. Supported by NIH GM069783.

Law of corresponding states for osmotic swelling of vesicles

As solute molecules permeate into a vesicle due to a concentration difference across its membrane, the vesicle swells through osmosis. The swelling can be divided into two stages: (a) an "ironing" stage, where the volume-to-area ratio of the vesicle increases without a significant change in its area; (b) a stretching stage, where the vesicle grows while remaining essentially spherical, until it ruptures. We show that the crossover between these two stages can be represented as a broadened continuous phase transition. Consequently, the swelling curves for different vesicles and different permeating solutes can be rescaled into a single, theoretically predicted, universal curve. Such a data collapse is demonstrated for giant unilamellar POPC vesicles, osmotically swollen due to the permeation of urea, glycerol, or ethylene glycol. We thereby gain a sensitive measurement of the solutes' membrane permeability coefficients, finding a concentration-independent coefficient for urea, while those of glycerol and ethylene glycol are found to increase with solute concentration. In addition, we use the width of the transition, as extracted from the data collapse, to infer the number of independent bending modes that affect the thermodynamics of the vesicle in the transition region.

Effects of Hidrophobic Surfactant Proteins SP-B and SP-C on the Mechanical Properties and Structural Stability of Phospholipid Bilayers

The respiratory surface is stabilized by pulmonary surfactant, a complex mixture of lipids and proteins, whose main function is reducing surface tension at the alveolar air-liquid interface in order to facilitate the work of breathing. It is composed by around 90% lipids and 8-10% specific proteins, including the hydrophobic polypeptides SP-B and SP-C. A combined action of both proteins is essential for a proper organization of functional membrane arrays in surfactant complexes.SP-B and SP-C have dramatical effects on membrane structure and dynamics. In the present study we compare some structural, mechanical and dynamical properties of model POPC vesicles in the absence and presence of them, either in their physiological combined proportion or each protein by itself. Structural effects caused by hydrophobic surfactant proteins were noticed both by optical microscopy of giant proteolipid vesicles and by electron microscopy of 100 nm diameter extruded vesicles. Also, impermeable POPC membranes became permeable when supplemented with any of these proteins. Significant differences were noticed between the effect of SP-B and SP-C on giant vesicles: suspensions containing only SP-B were stable but those containing only SP-C were quite dynamic, undergoing frequent fluctuations, reorganizations and ruptures as observed under the microscope. In order to investigate the physical explanation of these phenomena, mechanical studies have been carried out with giant phospholipid and proteolipid vesicles.The differences found between the effect of each protein separately and their physiological mixture support the concept that SP-B and SP-C mutually modulate their membrane-perturbing properties, which may be crucial for the structure, arrangement and dynamics of pulmonary surfactant membranes.

Thermodynamic Measurements of Bilayer Insertion of a Single Transmembrane Helix Chaperoned by Fluorinated Surfactants

Accurate determination of the free energy of transfer of a helical segment from an aqueous into a transmembrane (TM) conformation is essential for understanding and predicting the folding and stability of membrane proteins. Until recently, direct thermodynamically sound measurements of free energy of insertion of hydrophobic TM peptides were impossible due to peptide aggregation outside the lipid bilayer. Here, we overcome this problem by using fluorinated surfactants that are capable of preventing aggregation but, unlike detergents, do not themselves interact with the bilayer. We have applied the fluorescence correlation spectroscopy methodology to study surfactant-chaperoned insertion into preformed POPC (palmitoyloleoylphosphatidylcholine) vesicles of the two well-studied dye-labeled TM peptides of different lengths: WALP23 and WALP27. Extrapolation of the apparent free-energy values measured in the presence of surfactants to a zero surfactant concentration yielded free-energy values of −9.0±0.1 and −10.0±0.1 kcal/mol for insertion of WALP23 and WALP27, respectively. Circular dichroism measurements confirmed helical structure of peptides in lipid bilayer, in the presence of surfactants, and in aqueous mixtures of organic solvents. From a combination of thermodynamic and conformational measurements, we conclude that the partitioning of a four-residue L–A–L–A segment in the context of a continuous helical conformation from an aqueous environment into the hydrocarbon core of the membrane has a favorable free energy of 1 kcal/mol. Our measurements, combined with the predictions of two independent experimental hydrophobicity scales, indicate that the per-residue cost of transfer of the helical backbone from water to the hydrocarbon core of the lipid bilayer is unfavorable and is equal to +2.13±0.17 kcal/mol.

Unique Cholesterol Transport Behavior in Phosphoserine Vesicles: A Small Angle Neutron Scattering Study

Cholesterol is critical for various cellular functions; however its excess is toxic. Cholesterol levels are maintained by various cholesterol metabolic pathways which depend critically on Intracellular cholesterol transport. Cholesterol is not homogeneously distributed in cell with 60-70% of cellular cholesterol present in plasma membrane and only 0.01-0.5% of cellular cholesterol present in endoplasmic reticulum. Any disruption in cholesterol distribution disorder has been linked to diseases such as Niemann Pick TYPE-C and Alziemers. It has been suggested that variable affinity of cholesterol for different lipids compositions could be one of the possible reason for the uneven distribution of cholesterol within cell. Traditionally the cyclodextrine or cholesterol oxidases have been used to measure the relative affinity of cholesterol for a particular lipid composition. However, the possible disruption of lipid-cholesterol interactions due to these molecules is unknown. This present study employs small angle neutron scattering to measure the cholesterol inter- and intra-membrane transport rate in model lipid vesicle without employing cholesterol tags and molecules such as cyclodextrine or cholesterol oxidase. The diffusion behavior was compared between two POPC vesicles and two POPS vesicles. Interestingly cholesterol exchange kinetics follows a non-continuous Arrhenius behavior in POPS membranes as compare to linear behavior in POPC membranes. Further cholesterol exchange kinetics was compared from POPC to POPS vesicles and POPS to POPC vesicles. Interestingly equilibrium cholesterol distribution changes with both cholesterol concentration and temperature. To our surprise we found that even a small amount of cyclodextrine can significantly shift the equilibrium distribution of cholesterol between POPS and POPC vesicles. Overall this wok provides insight in to POPS-cholesterol interaction and potential role of POPS is regulating intracellular cholesterol transport.

Effect of Hydrophobic Surfactant Proteins SP-B and SP-C on the Permeability of Phospholipid Membranes

Pulmonary surfactant is a complex mixture of lipids and proteins whose main function is to reduce surface tension at the alveolar air-liquid interface in order to avoid alveolar collapse at the end of expiration and facilitate the work of breathing. It is composed by around 90% lipids and 8-10% specific proteins, including the hydrophobic SP-B and SP-C. In this study, we have analyzed the effect of hydrophobic surfactant proteins on the permeability of phospholipid membranes by two different approaches: fluorescence microscopy of giant vesicles (GV) and electroconduction in planar lipid membranes. The effect of surfactant proteins on the permeability of GV membranes was assessed under the microscope using the fluorescent water-soluble probes FM®1-43 and calcein. Membrane-sensitive FM®1-43 only labels the external leaflet of membranes, and calcein emits green fluorescence in aqueous media. Neither can permeate through pure lipid membranes. In the presence of physiological amounts of SP-B and SP-C, giant oligolamellar POPC vesicles incorporated FM®1-43 in every single membrane when added to the external medium and were also permeable to calcein. These results suggest the existence of direct connections between aqueous compartments of GV in the presence of these proteins. On the other hand, planar lipid membranes (PLM) have been widely used to study ionic permeation through phospholipid bilayers mediated by membrane proteins. Permeability of bilayers incorporating small amounts of hydrophobic surfactant proteins has been analyzed in PLMs prepared by the dual monolayer technique. Conductance and selectivity experiments as well as noise analysis of the signals were performed. All our measurements indicate the formation of channel-like structures with defined properties in terms of ionic conductance and selectivity. Possible implications of membrane-permeabilizing structures in the biological context of the pulmonary surfactant system will be discussed.

On the Origin of Multi-Exponential Kinetics in Peptide Binding to Phospholipid Vesicles

Binding kinetics of amphipathic peptides to phospholipid bilayer vesicles are generally well described by a single exponential function. However, closer inspection of the data sometimes reveals the presence of additional exponential phases. These usually depend on the exact experimental conditions and on the peptide studied, but are indicative of other processes that contribute to the binding kinetics. We investigated the binding of a model amphipathic peptide, Lysette-26, to unilamellar lipid vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a function of lipid and peptide concentration. Lysette-26 is a 22 residue peptide with the sequence IISTIGDLVKWIIDTVKWIIDTVNKFTKK. The peptide binds well to zwitterionic lipid bilayer membranes and forms an amphipathic α-helix when bound at the membrane-water interface. Peptide binding was measured through fluorescence energy transfer from the intrinsic tryptophan residue in the peptide to an acceptor fluorophore embedded in the membrane at low concentrations. A series of exact kinetic models was developed to describe the experimental data. We found that the kinetics of peptide binding to POPC vesicles depended strongly on peptide concentration in solution. The model that best described the experimental data included the presence of peptide monomers and aggregates in solution that bind independently and with different rate constants to the lipid vesicles. Peptide aggregation is favored at higher peptide concentrations, leading to the appearance of an additional exponential phase. Based on the kinetic models, other processes, such as peptide insertion or translocation across the bilayer, could be excluded as the source of the second exponential phase in the binding kinetics.

A Thermodynamic Approach to the Mechanism of Cell-Penetrating Peptides in Model Membranes

We report a first test of the hypothesis that the mechanism of antimicrobial, cytolytic, and amphipathic cell-penetrating peptides in model membranes is determined by the thermodynamics of insertion of the peptide into the lipid bilayer from the surface-associated state. Three peptides were designed with minimal mutations relative to the sequence of TP10W, the Y3W variant of transportan 10, which is a helical, amphipathic cell-penetrating peptide previously studied. Binding to 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) membranes and release of dye from those vesicles were assessed by stopped-flow fluorescence, and the secondary structure of the peptides on the membrane was determined by circular dichroism. The Gibbs energy of binding determined experimentally was in excellent agreement with that calculated using the Wimley-White interfacial hydrophobicity scale, taking into account the helical content of the membrane-associated peptide. Release of dye from POPC vesicles remained graded, as predicted by the hypothesis. More significantly, as the Gibbs energy of insertion into the bilayer became more unfavorable, which was estimated using the Wimley-White octanol hydrophobicity scale, dye release became slower, in quantitative agreement with the prediction.

Detection of Association and Fusion of Giant Vesicles Using a Fluorescence-Activated Cell Sorter

We have developed a method to evaluate the fusion process of giant vesicles using a fluorescence-activated cell sorter (FACS). Three fluorescent markers and FACS technology were used to evaluate the extent of association and fusion of giant vesicles. Two fluorescent markers encapsulated in different vesicle populations were used as association markers; when these vesicles associate, the two independent markers should be observed simultaneously in a single detection event. The quenched fluorescent marker and the dequencher, which were encapsulated in separate vesicle populations, were used as the fusion marker. When the internal aqueous solutions mix, the quenched marker is liberated by the dequencher and emits the third fluorescent signal. Although populations of pure POPC vesicles showed no detectable association or fusion, the same populations, oppositely charged by the exogenous addition of charged amphiphiles, showed up to 50% association and 30% fusion upon population analysis of 100,000 giant vesicles. Although a substantial fraction of the vesicles associated in response to a small amount of the charged amphiphiles (5% mole fraction compared to POPC alone), a larger amount of the charged amphiphiles (25%) was needed to induce vesicle fusion. The present methodology also revealed that the association and fusion of giant vesicles was dependent on size, with larger giant vesicles associating and fusing more frequently.

The structural parameters for antimicrobial activity, human epithelial cell cytotoxicity and killing mechanism of synthetic monomer and dimer analogues derived from hBD3 C-terminal region

Understanding the molecular mechanisms of antimicrobial peptide-membrane interactions is crucial in predicting the design of useful synthetic antimicrobial peptide analogues. Defensins are small (3-5 kDa) cysteine-rich cationic proteins which constitute the front line of host innate immunity. In this study, a series of eight 10 AA C-terminal analogues of hBD3 [sequence: RGRKXXRRKK, X = W, F, Y, V, L, I, H, C(Acm); net charge = +7, coded as W2, F2, Y2, V2, L2, I2, H2, and C2] and covalent V2-dimer [(RGRKVVRR)(2)KK] (18 AA, net charge = +11) were synthesized using solid phase peptide synthesis (SPPS) in Fmoc chemistry. Wild-type hBD3 was used as a control in all analyses. W2, V2, and especially Y2 showed high activity selectively against Gram-negative bacteria Pseudomonas aeruginosa in the concentration range of 4.3-9.7 microM. The covalent dimeric form of V2-monomer, V2-dimer, showed increased antibacterial killing compared to the monomeric form, V2-monomer. Cytotoxicity assays on a human conjunctival epithelial cell line (IOBA-NHC cells) showed that no change in viable cell number 24 h after constant exposure to all the eight peptide analogues even at concentrations up to 200 microg/ml. Fluorescence correlation spectroscopy (FCS) was used to study the interaction of these peptides against POPC vesicles (neutral; mammalian cell membrane mimic) and POPG vesicles (negatively charged; bacterial cell membrane mimic). Using FCS, significant aggregation and some leakage of Rhodamine dye were observed with POPG with Y2, W2 and V2 at the concentration of 5-10 mmicroM and no significant aggregation or disruption of vesicles was observed for all peptide analogues tested against POPC. V2-dimer induced more leakage and aggregation than the monomeric form. Overall, V2-dimer is the most effective antimicrobial peptide, with aggregation of POPG vesicles observed at concentrations as low as 1 microM. The concentration of 5-10 microM for Y2 from FCS correlated with the concentration of 5 microM (6.25 microg/ml), at which Y2 showed a cooperative increase in the activity. This suggests a structural transition of Y2 in the 2.5-5 microM concentration range resulting in the correlated increased antimicrobial activity. These results and the FCS together with previous NMR and molecular dynamics (MD) suggested that the charge density-based binding affinity, stable covalent dimerization, the ability to dimerize or even oligomerize and adopt a well-defined structure are important physicochemical properties distinguishing more effective cationic antimicrobial peptides.

Calix[4]pyrrole-based anion transporters with tuneable transport properties

Three new bis-1,2,3-triazole strapped calix[4]pyrroles have been prepared via'click' chemistry and their anion complexation and lipid bilayer transport properties studied by a combination of single crystal X-ray diffraction studies, (1)H NMR titration techniques, isothermal titration calorimetry and lipid bilayer anion transport studies in POPC vesicles. Bilayer transport efficiency for transmembrane chloride transport was found to directly depend on the length of the alkyl chain present in the bis-triazole strap.

Different Scenarios of Membrane Permeabilization by Bacterial Lipopeptides

The fungicidal activity of Bacillus subtilis QST 713, based mainly on the production of fengycin (FE, including several agrastatins and plipastatins), surfactin (SF), and iturin (IT) lipopeptides, has been utilized for a highly effective and environmentally safe protection of crops against a variety of pathogens. Here we use a new assay, lifetime-based calcein leakage, to study their activity, selectivity, and mechanism of membrane permeabilization. SF permeabilizes monounsaturated POPC vesicles essentially like an extremely potent detergent: It causes graded leakage starting at about Re = 0.05 peptides/lipid in the membrane and releases all dye already below the concentration required for lysis to micelles. FE shows a totally different behaviour; leakage is all-or-none and reaches a plateau after opening of 15% of the vesicles; further progress of leakage is very weak up to high peptide concentrations. Further information is obtained from ITC, fluorescence spectroscopy, light scattering, and cryo-TEM. We explain this very unusual behaviour of FE analogously to the phenomenon of detergent-resistant membranes, although no such resistance has been described so far for a cholesterol-free membrane of an unsaturated lipid. These surprising findings have major consequences for the biological activity and possible technical applications of the lipopeptides.

Structural Determinants of Antimicrobial Activity and Biocompatibility in Membrane-Disrupting Methacrylamide Random Copolymers

Low molecular weight random copolymers bearing protonated primary amine groups and hydrophobic alkyl groups in the side chains were synthesized and their activities against E. coli , S. aureus , human red blood cells, and human epithelial carcinoma cells (HEp-2) were quantified. The mole fraction of alkyl side chains in the copolymers (f(alkyl)) and the length of the alkyl chains were major determinants of the activities. Against E. coli cells, activity was diminished as f(alkyl) was increased from 0 to about 0.2, but was then enhanced dramatically as f(alkyl) was increased further. Activity against S. aureus was diminished continually with increasing f(alkyl). The cytotoxicity to human epithelial carcinoma cells also decreased with increasing f(alkyl). Conversely, hemolytic activity showed monotonic enhancement with increasing f(alkyl). The cationic homopolymer (f(alkyl) = 0) completely inhibited S. aureus growth at 3 microM (10.2 microg/mL) and completely inhibited metabolic activity in HEp-2 cells at 10 microM (34 microg/mL), although it did not induce any detectable hemolysis up to 645 microM (2000 microg/mL). Polymer-induced dye leakage from liposomes provided a biophysical basis for understanding the factors which modulate the polymer-membrane interactions. Disruption of Zwitterionic POPC vesicles induced by the copolymers was enhanced as f(alkyl) increased, following trends similar to the hemolytic activity data. The ability of the polymers to permeabilize vesicles of POPE/POPG and DOPG/Lysyl-DOPG/CL displayed trends similar to trends in their activities against E. coli and S. aureus , respectively. This was interpreted as evidence that the antimicrobial mechanism employed by the polymers involves disruption of bacterial cell membranes. An investigation of leakage kinetics revealed that the cationic homopolymer induced a gradual release of contents from POPE/POPG and DOPG/Lysyl-DOPG/CL vesicles, while the more hydrophobic copolymers induced rapid dye efflux. The results are interpreted as evidence that the cationic homopolymer and hydrophobic copolymers in this study exert their antimicrobial action by fundamentally different mechanisms of membrane disruption.

Preparation of reconstituted acetylcholine receptor membranes suitable for AFM imaging of lipid–protein interactions

The nicotinic acetylcholine receptor (nAChR) has been reconstituted in POPC vesicles at high lipid–protein (L/P) ratios for the preparation of supported lipid bilayers with a low protein density for studies of protein–lipid interactions using atomic force microscopy (AFM). Initial reconstitutions using a standard dialysis method with bulk L/P ratios ranging from 20:1 to 100:1 (w/w) gave heterogeneous samples that contained both empty vesicles and proteoliposomes with a range of L/P ratios. This is problematic because empty vesicles adsorb and rupture to form bilayer patches more rapidly than do protein-rich vesicles, resulting in the loss of protein during sample washing. Although it was not possible to find reconstitution conditions that gave homogeneous populations of vesicles with high L/P ratios, an additional freeze–thaw cycle immediately after dialysis did reproducibly yield a fraction of proteoliposomes with L/P ratios above 100:1. These proteoliposomes were separated by sucrose gradient centrifugation and used to prepare supported bilayers with well-separated individual receptors and minimal adsorbed proteoliposomes. AFM images of such samples showed many small features protruding from the bilayer surface. These features range in height from 1 to 5 nm, consistent with the smaller intracellular domain of the protein exposed, and have lateral dimensions consistent with an individual receptor. Some bilayers with reconstituted protein also had a small fraction of higher features that are assigned to nAChR with the larger extracellular domain exposed and showed evidence for aggregation to give dimers or small oligomers. This work demonstrates the importance of using highly purified reconstituted membranes with uniform lipid–protein ratios for AFM studies of integral membrane protein–lipid interactions.

Fluctuation dynamics of spherical vesicles: Frustration of regular bulk dissipation into subdiffusive relaxation

Spherical lipid vesicles obtained by the extrusion method are nonequilibrium membrane structures more curved than the zero spontaneous curvature equilibrium state of the bilayer. Furthermore, these structures are quite rigid as compared to spontaneous vesicles or microemulsion droplets made of soluble surfactants. The dynamical description of the shape fluctuations derived by Milner and Safran (MS) [Phys. Rev. A 36, 4371 (1987)], which is based on the elastic Helfrich energy referred to the equilibrium state, could be misleading in these cases. In the present contribution, shape fluctuations of unilamellar palmitoyl-oleyl-phosphocholine (POPC) vesicles (radius Rh<or=100 nm ) prepared by extrusion are studied by means of neutron spin echo (NSE) in combination with dynamic light scattering (DLS). The relaxation of the fluctuation modes is inferred from the DLS autocorrelation functions and from the intermediate NSE scattering functions measured for several different values of the wave vector. The observed relaxations are compatible with a stretched-exponential decay rather than the single-exponential behavior. Dynamical frustration of the bulk dissipation mechanism in the way described by Zilman and Granek (ZG) for weak amplitude fluctuations [Phys. Rev. Lett. 77, 4788 (1996)] is invoked as a plausible scenario for explaining the subdiffusive nonexponential relaxations experimentally observed. The combined analysis of NSE and DLS data allows a calculation of the bending elastic constant of POPC vesicles kappa=19+/-2 kBT , in excellent agreement with literature data. The ZG approach is revealed as the adequate extension of the MS theory to describe fluctuation dynamics of rigid membranes.

Characterization of Phospholipase Cγ Enzymes with Gain-of-Function Mutations

Phospholipase C gamma isozymes (PLC gamma 1 and PLC gamma 2) have a crucial role in the regulation of a variety of cellular functions. Both enzymes have also been implicated in signaling events underlying aberrant cellular responses. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we have recently identified single point mutations in murine PLC gamma 2 that lead to spontaneous inflammation and autoimmunity. Here we describe further, mechanistic characterization of two gain-of-function mutations, D993G and Y495C, designated as ALI5 and ALI14. The residue Asp-993, mutated in ALI5, is a conserved residue in the catalytic domain of PLC enzymes. Analysis of PLC gamma 1 and PLC gamma 2 with point mutations of this residue showed that removal of the negative charge enhanced PLC activity in response to EGF stimulation or activation by Rac. Measurements of PLC activity in vitro and analysis of membrane binding have suggested that ALI5-type mutations facilitate membrane interactions without compromising substrate binding and hydrolysis. The residue mutated in ALI14 (Tyr-495) is within the spPH domain. Replacement of this residue had no effect on folding of the domain and enhanced Rac activation of PLC gamma 2 without increasing Rac binding. Importantly, the activation of the ALI14-PLC gamma 2 and corresponding PLC gamma 1 variants was enhanced in response to EGF stimulation and bypassed the requirement for phosphorylation of critical tyrosine residues. ALI5- and ALI14-type mutations affected basal activity only slightly; however, their combination resulted in a constitutively active PLC. Based on these data, we suggest that each mutation could compromise auto-inhibition in the inactive PLC, facilitating the activation process; in addition, ALI5-type mutations could enhance membrane interaction in the activated state.