dioleoyl-phosphatidylethanolamine and 1-2-dioleoyl-sn-glycero-3-phosphoglycerol

In cells, the breakdown of arginine to ornithine and ammonium ion plus carbon dioxide is coupled to the generation of metabolic energy in the form of ATP. The arginine breakdown pathway is minimally composed of arginine deiminase, ornithine transcarbamoylase, carbamate kinase, and an arginine/ornithine antiporter; ammonia and carbon dioxide most likely diffuse passively across the membrane. The genes for the enzymes and transporter have been cloned and expressed, and the proteins have been purified from Lactococcus lactis IL1403 and incorporated into lipid vesicles for sustained production of ATP. Here, we study the kinetic parameters and biochemical properties of the individual enzymes and the antiporter, and we determine how the physicochemical conditions, effector composition, and effector concentration affect the enzymes. We report the K

Topics: Adenosine Triphosphate; Amino Acid Transport Systems; Ammonia; Antiporters; Arginine; Bacterial Proteins; Carbon Dioxide; Energy Metabolism; Gene Expression Regulation, Bacterial; Hydrolases; Kinetics; Lactococcus lactis; Liposomes; Ornithine; Ornithine Carbamoyltransferase; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphotransferases (Carboxyl Group Acceptor); Recombinant Proteins

Topics: Bacteria; Cell Membrane; Membranes, Artificial; Oxidation-Reduction; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Photochemical Processes; Tolonium Chloride

Novel polymers containing quaternary functional groups, with and without (control copolymer) PEG side chains, were synthesized and characterized for their ability to lyse the phospholipid membranes of liposome vesicles. Calcein loaded unilamellar vesicles composed of 1,2-dioleoyl- sn-glycero-3-phosphatidylcholine (DOPC) were used to mimic red-blood cell membranes, and a 80:20 (mol/mol) mixture of 1,2-dioleoyl- sn-glycero-3-phosphatidyl ethanolamine (DOPE) and 1,2-dioleoyl- sn- glycero-3-[phospho- rac-(1-glycerol)] (DOPG) was used to mimic the outer cell-membrane of the gram-negative bacteria, E. coli. For DOPE/DOPG = 80:20 (mol/mol) liposome vesicles, the PEG bottle brush copolymer caused leakage of the encapsulated Calcein dye, whereas the control copolymer did not cause any leakage. Both the bottle brush copolymer and the copolymer without PEG side chains had no effect on the zwitterionic DOPC liposome vesicles indicating that the RBC membrane composition is not disrupted by either copolymer architecture. The PEG bottle brush copolymer did not affect the colloidal size of the DOPE/DOPG = 80:20 (mol/mol) liposome vesicles, but on the addition of Triton-X 100, the vesicles disappeared. This provided evidence that the dye leakage was caused by compromising the integrity of the vesicle membrane by the bottle brush polymer architecture. Such partial disruption was preceded by the entropic templating of lipid membranes by the PEG side chains of the bottle brush copolymer. By careful comparison with non-PEGylated cationic polymers, Quart, the importance of PEG side chains in the membrane disrupting activity of the PEGylated cationic polymer, QPEG, was demonstrated. This finding itself is interesting and can contribute to the expansion of the design of membrane disrupting materials.

Topics: Coloring Agents; Fluoresceins; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Polyamines; Polyethylene Glycols; Unilamellar Liposomes

Protein import into the

Topics: Amino Acid Sequence; Binding Sites; Biomimetic Materials; Cell Fractionation; Cholesterol; Gene Expression; Hydrophobic and Hydrophilic Interactions; Leishmania donovani; Membrane Fluidity; Microbodies; Peroxisome-Targeting Signal 1 Receptor; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphatidylinositols; Protein Binding; Protein Conformation, alpha-Helical; Protein Interaction Domains and Motifs; Protozoan Proteins; Recombinant Proteins; Sequence Alignment; Unilamellar Liposomes

Antimicrobial peptides (AMPs) inactivate microorganisms by forming transmembrane pores in a cell membrane through adsorption and aggregation. Energetics of addition of an AMP to a transmembrane pore is important for evaluation of its formation and growth. Such information is essential for the characterization of pore forming ability of peptides in cell membranes. This study quantifies the potential of mean force through molecular dynamics (MD) simulation for the addition of melittin, a naturally occurring AMP, into a DOPC/DOPG mixed bilayer, a mimic of bacterial membrane, for different extents of insertion into either a bilayer or a pore consisting of three to six transmembrane peptides. The energy barrier for insertion of a melittin molecule into the bilayer was highest in the absence of transmembrane peptides and decreased for the number of transmembrane peptides from three to six, eventually approaching zero. The decrease in free energy for complete insertion of peptide was found to be higher for larger pore size. Water channel formation occurred only for insertion into pores consisting of three or more transmembrane peptides with the radius of water channel being larger for a larger number of transmembrane peptides. The structure of the pore was found to be paraboloid. The estimated free energy barrier for insertion of melittin into an ideal paraboloid pore accounting for different intermolecular interactions was consistent with MD simulation results. The results reported in this manuscript will be useful for the development of a model for nucleation of pores and a rational methodology for selection of synthetic antimicrobial peptides.

Topics: Amino Acid Sequence; Lipid Bilayers; Melitten; Models, Chemical; Molecular Dynamics Simulation; Phosphatidylethanolamines; Phosphatidylglycerols; Physical Phenomena

Antibiotic resistance is a growing concern in medicine and raises the need to develop and design new drug molecules that can efficiently inhibit bacterial replication. Spurring the passive uptake of the drug molecules is an obvious solution. However our limited understanding of drug-membrane interactions due to the presence of an overwhelming variety of lipids constituting cellular membranes and the lack of facile tools to probe the bio-physical interactions between drugs and lipids imposes a major challenge towards developing new drug molecules that can enter the cell via passive diffusion. Here, we used a label-free micro-fluidic platform combined with giant unilamellar lipid vesicles to investigate the permeability of membranes containing mixtures of DOPE and DOPG in DOPC, leading to a label-free measurement of passive membrane-permeability of autofluorescent antibiotics. A fluoroquinolone drug, norfloxacin was used as a case study. Our results indicate that the diffusion of norfloxacin is strongly dependent on the lipid composition which is not expected from the traditional octanol-lipid partition co-efficient assay. The anionic lipid, DOPG, slows the diffusion process whereas the diffusion across liposomes containing DOPE increases with higher DOPE concentration. Our findings emphasise the need to investigate drug-membrane interactions with focus on the specificity of drugs to lipids for efficient drug delivery, drug encapsulation and targeted drug-delivery.

Topics: Anti-Bacterial Agents; Kinetics; Lab-On-A-Chip Devices; Lipid Bilayers; Norfloxacin; Permeability; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Static Electricity; Unilamellar Liposomes

The water-in-oil droplet bilayer is a simple and useful lipid bilayer system for membrane transport analysis. The droplet interface bilayer is readily formed by the contact of two water-in-oil droplets enwrapped by a phospholipid monolayer. However, the size of individual droplets with femtoliter volumes in a high-throughput manner is difficult to control, resulting in low sensitivity and throughput of membrane transport analysis. To overcome this drawback, in this study, we developed a novel micro-device in which a large number of droplet interface bilayers (>500) are formed at a time by using femtoliter-sized droplet arrays immobilized on a hydrophobic/hydrophilic substrate. The droplet volume was controllable from 3.5 to 350 fL by changing the hydrophobic/hydrophilic pattern on the device, allowing high-throughput analysis of membrane transport mechanisms including membrane permeability to solutes (e.g., ions or small molecules) with or without the aid of transport proteins. Thus, this novel platform broadens the versatility of water-in-oil droplet bilayers and will pave the way for novel analytical and pharmacological applications such as drug screening.

Topics: Algorithms; Bacterial Toxins; Biological Transport; Calcium Chloride; Coloring Agents; Equipment Design; Hemolysin Proteins; High-Throughput Screening Assays; Hydrophobic and Hydrophilic Interactions; Lipid Bilayers; Microarray Analysis; Models, Biological; Permeability; Phosphatidylethanolamines; Phosphatidylglycerols

Determining the mechanism of action of antimicrobial peptides (AMPs) is critical if they are to be developed into the clinical setting. In recent years high resolution techniques such as atomic force microscopy (AFM) have increasingly been utilised to determine AMP mechanism of action on planar lipid bilayers and live bacteria. Here we present the biophysical characterisation of a prototypical AMP from the venom of the North African scorpion Scorpio maurus palmatus termed Smp24. Smp24 is an amphipathic helical peptide containing 24 residues with a charge of +3 and exhibits both antimicrobial and cytotoxic activity and we aim to elucidate the mechanism of action of this peptide on both membrane systems. Using AFM, quartz crystal microbalance-dissipation (QCM-D) and liposomal leakage assays the effect of Smp24 on prototypical synthetic prokaryotic (DOPG:DOPC) and eukaryotic (DOPE:DOPC) membranes has been determined. Our data points to a toroidal pore mechanism against the prokaryotic like membrane whilst the formation of hexagonal phase non-lamellar phase structures is seen in eukaryotic like membrane. Also, phase segregation is observed against the eukaryotic membrane and this study provides direct evidence of the same peptide having multiple mechanisms of action depending on the membrane lipid composition.

Topics: Animals; Antimicrobial Cationic Peptides; Lipid Bilayers; Liposomes; Molecular Mimicry; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Protein Conformation, alpha-Helical; Scorpion Venoms; Scorpions; Static Electricity

Whereas many membrane-destabilization modes have been suggested for membrane-spanning antimicrobial peptides (AMPs), few are available for those too short to span membrane thickness. Here we show that ORB-1, a 15-residue disulfide-bridged AMP that is only ∼20 Å long even when fully stretched like a hairpin, may act by inducing small anion-selective transmembrane "holes" of negative mean curvature. In model membranes of Gram-negative bacteria, ORB-1 induces chloride transmembrane transport and formation of transmembrane channels of negative mean curvature, whereas the inactive analogue, ORB-N, does not, suggesting a correlation between antibacterial activity and ability to induce transmembrane channels. Given that ORB-N is the C-terminus amidated form of ORB-1, our results further suggest that formation of membrane-spanning dimers may be required to initiate the observed channel induction. Moreover, ORB-1 renders model bacterial membranes permeable to anions with effective hydration diameters of <1 nm (e.g., Cl(-) and NO3(-)), but not cations of similar sizes (e.g., H3O(+)), indicative of anion-selective transmembrane channels with an effective inner diameter of ≤1 nm. In addition, negative-intrinsic-curvature (NIC) lipids such as phosphoethanolamine (PE) may facilitate the membrane-destabilization process of ORB-1. Our findings may expand current understandings on how AMPs destabilize membranes and facilitate the pharmaceutical development of ORB-1.

Topics: Anions; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Cell Membrane; Dimerization; Escherichia coli; Hemolysis; Ion Channels; Ion Transport; Models, Biological; Phosphatidylethanolamines; Phosphatidylglycerols; Staphylococcus aureus; Unilamellar Liposomes

Although the importance of a SNARE complex in neurotransmitter release is widely accepted, there exist different views on how the complex promotes fusion. One hypothesis is that the SNARE complex's ability to bring membranes into contact is sufficient for fusion, another points to possible roles of juxtamembrane regions (JMRs) and transmembrane domains (TMDs) in catalyzing lipid rearrangement, and another notes the complex's presumed ability to bend membranes near the point of contact. Here, we performed experiments with highly curved vesicles brought into contact using low concentrations of polyethylene glycol (PEG) to investigate the influence of the synaptobrevin (SB) TMD with an attached JMR (SB-JMR-TMD) on the rates of stalk and pore formation during vesicle fusion. SB-JMR-TMD enhanced the rates of stalk and fusion pore (FP) formation in a sharply sigmoidal fashion. We observed an optimal influence at an average of three peptides per vesicle, but only with phosphatidylserine (PS)-containing vesicles. Approximately three SB-JMR-TMDs per vesicle optimally ordered the bilayer interior and excluded water in a similar sigmoidal fashion. The catalytic influences of hexadecane and SB-JMR-TMD on fusion kinetics showed little in common, suggesting different mechanisms. Both kinetic and membrane structure measurements support the hypotheses that SB-JMR-TMD 1) catalyzes initial intermediate formation as a result of its basic JMR disrupting ordered interbilayer water and permitting closer interbilayer approach, and 2) catalyzes pore formation by forming a membrane-spanning complex that increases curvature stress at the circumference of the hemifused diaphragm of the prepore intermediate state.

Topics: Catalysis; Exocytosis; Kinetics; Lipid Bilayers; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphatidylserines; Polyethylene Glycols; R-SNARE Proteins; Synaptic Vesicles; Thermodynamics; Unilamellar Liposomes

The phospholipid composition that surrounds a membrane protein is critical to maintain its structural integrity and, consequently, its functional properties. To understand better this in the present work we have performed FRET measurements between the single tryptophan residue of a lactose permease Escherichia coli mutant (single-W151/C154G LacY) and pyrene-labeled phospholipids (Pyr-PE and Pyr-PG) at 37 degrees C. We have reconstituted this LacY mutant in proteoliposomes formed with heteroacid phospholipids, POPE and POPG, and homoacid phospholipids DOPE and DPPE, resembling the same PE/PG proportion found in the E. coli inner membrane (3:1, mol/mol). A theoretical model has been fitted to the experimental data. In the POPE/POPG system, quantitative model calculations show accordance with the experimental values that requires an annular region composed of approximately approximately 90 mol% PE. The experimental FRET efficiencies for the gel/fluid phase-separated DOPE/POPG system indicate a higher presence of PG in the annular region, from which it can be concluded that LacY shows clear preference for the fluid phase. Similar conclusions are obtained from analysis of excimer-to-monomer (E/M) pyrene ratios. To test the effects of this on cardiolipin (CL) on the annular region, myristoyl-CL and oleoyl-CL were incorporated in the biomimetic POPE/POPG matrix. The experimental FRET efficiency values, slightly larger for Pyr-PE than for Pyr-PG, suggest that CL displaces POPE and, more extensively, POPG from the annular region of LacY. Model fitting indicates that CL enrichment in the annular layer is, in fact, solely produced by replacing PG and that myristoyl-CL is not able to displace PE in the same way that oleoyl-CL does. One of the conclusions of this work is the fact that LacY inserts preferentially in fluid phases of membranes.

Topics: Cardiolipins; Fluorescence Resonance Energy Transfer; Lipid Bilayers; Membrane Transport Proteins; Phosphatidylethanolamines; Phosphatidylglycerols

The effect of hydrophobic peptides on the lipid phase behavior of an aqueous dispersion of dioleoylphosphatidylethanolamine and dioleoylphosphatidylglycerol (7:3 molar ratio) was studied by (31)P NMR spectroscopy. The peptides (WALPn peptides, where n is the total number of amino acid residues) are designed as models for transmembrane parts of integral membrane proteins and consist of a hydrophobic sequence of alternating leucines and alanines, of variable length, that is flanked on both ends by tryptophans. The pure lipid dispersion was shown to undergo a lamellar-to-isotropic phase transition at approximately 60 degrees C. Small-angle x-ray scattering showed that at a lower water content a cubic phase belonging to the space group Pn3m is formed, suggesting also that the isotropic phase in the lipid dispersion represents a cubic liquid crystalline phase. It was found that the WALP peptides very efficiently promote formation of nonlamellar phases in this lipid system. At a peptide-to-lipid (P/L) molar ratio of 1:1000, the shortest peptide used, WALP16, lowered the lamellar-to-isotropic phase transition by approximately 15 degrees C. This effect was less for longer peptides. For all of the WALP peptides used, an increase in peptide concentration led to a further lowering of the phase transition temperature. At the highest P/L ratio (1:25) studied, WALP16 induced a reversed hexagonal liquid crystalline (H(II)) phase, while the longer peptides still promoted the formation of an isotropic phase. Peptides with a hydrophobic length larger than the bilayer thickness were found to be unable to inhibit formation of the isotropic phase. The results are discussed in terms of mismatch between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer and its consequences for lipid-protein interactions in membranes.

Topics: Amino Acid Sequence; Biophysical Phenomena; Biophysics; Escherichia coli; Magnetic Resonance Spectroscopy; Membrane Lipids; Membranes, Artificial; Peptides; Phosphatidylethanolamines; Phosphatidylglycerols; X-Ray Diffraction

The Pf3 major coat protein of the Pf3 bacteriophage is stored in the inner membrane of the infected cell during the reproductive cycle. The protein consists of 44 amino acids, and contains an acidic amphipathic N-terminal domain, a hydrophobic domain, and a short basic C-terminal domain. The mainly alpha-helical membrane-bound protein traverses the membrane once, leaving the C-terminus in the cytoplasm and the N-terminus in the periplasm. A cysteine-scanning approach was followed to measure which part of the membrane-bound Pf3 protein is inside or outside the membrane. In this approach, the fluorescence probe N-[(iodoacetyl)amino]ethyl-1-sulfonaphthylamine (IAEDANS) was attached to single-cysteine mutants of the Pf3 coat protein. The labeled mutant coat proteins were reconstituted into the phospholipid DOPC/DOPG (80/20 molar ratio) and DOPE/DOPG (80/20 molar ratio) model membranes. We subsequently studied the fluorescence characteristics at the different positions in the protein. We measured the local polarity of the environment of the probe, as well as the accessibility of the probe to the fluorescence quencher acrylamide. The results of this study show a single membrane-spanning protein with both the C- and N-termini remaining close to the surface of the membrane. A nearly identical result was seen previously for the membrane-bound M13 coat protein. On the basis of a comparison between the results from both studies, we suggest an "L-shaped" membrane-bound model for the Pf3 coat protein. DOPE-containing model membranes revealed a higher polarity, and quenching efficiency at the membrane/water interface. Furthermore, from the outside to the inside of the membrane, a steeper polarity gradient was measured at the PE/PG interface as compared to the PC/PG interface. These results suggest a thinner interface for DOPE/DOPG than for DOPC/DOPG membranes.

Topics: Amino Acid Sequence; Bacteriophage M13; Capsid; Capsid Proteins; Cysteine; Inovirus; Membrane Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Naphthalenesulfonates; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phospholipids; Pseudomonas aeruginosa; Pseudomonas Phages; Spectrometry, Fluorescence; Virus Assembly

The effect of nonlamellar-prone lipids, diacylglycerol (DG) and phosphatidylethanolamine (PE), on the ATPase activity of SecA was examined. When Escherichia coli PE of the standard vesicles composed of 60 mol% of this lipid and 40 mol% of dioleoylphosphatidylglycerol (DOPG) is gradually replaced with either dioleoylglycerol (DOG) or dioeloyl PE (DOPE), the ATPase activity of SecA present together increased appreciably. On the other hand, when E. coli PE of the standard vesicles was replaced with DOG analogs, the SecA ATPase activity decreased slightly, and when replaced with phosphatidylcholine the decrease in the ATPase activity was more appreciable. When DOPE or E. coli PE was added to PC vesicles, the SecA ATPase activity was enhanced only slightly, suggesting that the hexagonal II structure per se is not important for the ATPase activity increase. It was observed that DOG induced phase separation of PG, and the lamellar-hexagonal II (L-HII) transition temperature of vesicles decreased by about 10 degreesC. The DOG analogs had no effect on these properties, suggesting the importance of the phase separation of PG and the decrease of L-HII transition temperature of lipid bilayers to the SecA ATPase activity. The phase separation of PG by Ca2+ also brought about increased ATPase activity of SecA, underlining the importance of phase separation of PG for the enzyme activity. The incorporation of DOG or DOPE in the vesicle also increased the amount of SecA bound to model membranes and the extent of SecA penetration into the membrane. Studies with vesicles without SecA showed increased exposure of hydrophobic acyl chains when the DOG was present. Taken together, these observations suggest that the phase separation of PG and/or the bilayer penetration of SecA are mainly responsible for the enhanced SecA-vesicle interaction with concomitant increase in SecA ATPase activity.

Topics: Adenosine Triphosphatases; Bacterial Proteins; Diglycerides; Escherichia coli Proteins; Lipid Metabolism; Membrane Transport Proteins; Membranes, Artificial; Models, Biological; Phosphatidylethanolamines; Phosphatidylglycerols; SEC Translocation Channels; SecA Proteins; Spectrometry, Fluorescence; Temperature