1-palmitoyl-2-oleoylphosphatidylcholine and 1-2-dioleoylphosphatidylserine

Cardiotoxins (CTXs) are single polypeptide chain consisting of 59-62 amino acids with four disulfide bridges and globular proteins of simple β-sheet folds. The CTXs are one of principal toxic components causing haemolysis and damaging various cells and belong to three-finger toxin (TFT) superfamily of snake venoms. However, there is no natural or synthetic small molecular inhibitor to the protein toxins to date. In the present study, modes of interaction of cardiotoxin 1 (CTX1) from Indian cobra (Naja naja) with heterogeneous erythrocyte membrane (EM) model system have been extensively examined by using all-atom molecular dynamics (MD) simulations in near physiological conditions and comprehensive analyses of the MD data revealed two distinct principal regions ('head groove' and 'loop groove') of the protein toxin for establishing structural interactions with the EM system. Moreover, combined analyses of data from high-throughput virtual screening of NCI small molecular database, in vitro haemolytic assays for top-hits of the chemical compounds against crude venom of Naja naja and as well CTXs purified from the venom and pharmacokinetic examinations on the chemical compounds retarding haemolytic activities of CTXs suggested that Etidronic acid and Zoledronic acid are promising prototypic chemical inhibitors to CTXs of snake venoms.

Topics: Amino Acid Sequence; Animals; Antidotes; Cholesterol; Cobra Cardiotoxin Proteins; Diphosphonates; Disulfides; Elapid Venoms; Elapidae; Erythrocyte Membrane; Etidronic Acid; Hemolysis; High-Throughput Screening Assays; Humans; Imidazoles; Molecular Dynamics Simulation; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Protein Domains; Protein Structure, Secondary; Small Molecule Libraries; Structure-Activity Relationship; User-Computer Interface; Zoledronic Acid

The SNAREs SNAP25 and SNAP23 are proteins that are initially cytosolic after translation, but then become stably attached to the cell membrane through palmitoylation of cysteine residues. For palmitoylation to occur, membrane association is a prerequisite, but it is unclear which motif may increase the affinities of the proteins for the target membrane. In experiments with rat neuroendocrine cells, we find that a few basic amino acids in the cysteine-rich region of SNAP25 and SNAP23 are essential for plasma membrane targeting. Reconstitution of membrane-protein binding in a liposome assay shows that the mechanism involves protein electrostatics between basic amino acid residues and acidic lipids such as phosphoinositides that play a primary role in these interactions. Hence, we identify an electrostatic anchoring mechanism underlying initial plasma membrane contact by SNARE proteins, which subsequently become palmitoylated at the plasma membrane.

Topics: Amino Acid Motifs; Animals; Binding Sites; Cell Membrane; Cloning, Molecular; Escherichia coli; Gene Expression; Liposomes; Lipoylation; PC12 Cells; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Plasmids; Protein Binding; Protein Processing, Post-Translational; Protein Transport; Rats; Recombinant Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Static Electricity; Synaptosomal-Associated Protein 25; Vesicular Transport Proteins

Here we introduce ApoE-based nanolipoprotein particle (NLP)-a soluble, discoidal bilayer mimetic of ∼23 nm in diameter, as fusion partners to study the dynamics of fusion pores induced by SNARE proteins. Using in vitro lipid mixing and content release assays, we report that NLPs reconstituted with synaptic v-SNARE VAMP2 (vNLP) fuse with liposomes containing the cognate t-SNARE (Syntaxin1/SNAP25) partner, with the resulting fusion pore opening directly to the external buffer. Efflux of encapsulated fluorescent dextrans of different sizes show that unlike the smaller nanodiscs, these larger NLPs accommodate the expansion of the fusion pore to at least ∼9 nm, and dithionite quenching of fluorescent lipid introduced in vNLP confirms that the NLP fusion pores are short-lived and eventually reseal. The NLPs also have capacity to accommodate larger number of proteins and using vNLPs with defined number of VAMP2 protein, including physiologically relevant copy numbers, we find that 3-4 copies of VAMP2 (minimum 2 per face) are required to keep a nascent fusion pore open, and the SNARE proteins act cooperatively to dilate the nascent fusion pore.

Topics: Apolipoproteins E; Calcium; Cholesterol; Dextrans; Dimyristoylphosphatidylcholine; Dithionite; Fluorescent Dyes; Liposomes; Membrane Fusion; Nanoparticles; Particle Size; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphatidylserines; Synaptosomal-Associated Protein 25; Syntaxin 1; Vesicle-Associated Membrane Protein 2

Supported phospholipid membrane structures on cationic organic polymer beads were prepared using mixtures of dioleoylphosphatidylserine (PS) and egg yolk phosphatidylcholine (PC). Confocal fluorescence microscopic observations using a fluorescent membrane probe (N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine) revealed that the phospholipid molecules in the PS/PC-bead complexes were along the outer surface of the beads, but not inside the beads. The anionic PS on the most outer surface of the PS/PC-bead complexes was responsible for the binding of a positively charged macromolecule, rhodamine isothiocyanate dextran (M(w) 70,000) by electrostatic attractive forces. The fluidity of the membranes in the PS/PC-bead complexes was investigated by the fluorescence recovery after a photobleaching technique. The lateral diffusion coefficients (D) for the PS/PC-bead complexes were one-half or less than that for 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles without solid supporting materials. Such a constrain of the phospholipid bilayer membrane in the complexes appeared to be due to its immobilization on the cationic polymer bead by electrostatic attractive forces between the PS and ammonium group on the surface of the bead. The D values for the complexes were dependent on the phospholipid composition; the PS(25 mol%)/PC(75 mol%)-bead complex produced a more fluid membrane than the PS(50 mol%)/PC(50 mol%)-bead one. Thus, the fluidity of the phospholipid bilayer membranes formed on the cationic polymer beads was significantly affected by the anionic phospholipid fraction used for the preparation of the complexes.

Topics: Dextrans; Fluorescence Recovery After Photobleaching; Fluorescent Dyes; Lipid Bilayers; Membrane Fluidity; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Polymers; Rhodamines; Spectrometry, Fluorescence; Static Electricity; Unilamellar Liposomes

SNARE proteins play a critical role in intracellular membrane fusion by forming tight complexes that bring two membranes together and involve sequences called SNARE motifs. These motifs have a high tendency to form amphipathic coiled-coils that assemble into four-helix bundles, and often precede transmembrane regions. NMR studies in dodecylphosphocholine (DPC) micelles suggested that the N-terminal half of the SNARE motif from the neuronal SNARE synaptobrevin binds to membranes, which appeared to contradict previous biophysical studies of synaptobrevin in liposomes. NMR analyses of synaptobrevin reconstituted into nanodiscs and into liposomes now show that most of its SNARE motif, except for the basic C terminus, is highly flexible, exhibiting cross-peak patterns and transverse relaxation rates that are very similar to those observed in solution. Considering the proximity to the bilayer imposed by membrane anchoring, our data show that most of the synaptobrevin SNARE motif has a remarkable reluctance to bind membranes. This conclusion is further supported by NMR experiments showing that the soluble synaptobrevin SNARE motif does not bind to liposomes, even though it does bind to DPC micelles. These results show that nanodiscs provide a much better membrane model than DPC micelles in this system, and that most of the SNARE motif of membrane-anchored synaptobrevin is accessible for SNARE complex formation. We propose that the charge and hydrophobicity of SNARE motifs is optimized to enable formation of highly stable SNARE complexes while at the same time avoiding membrane binding, which could hinder SNARE complex assembly.

Topics: Amino Acid Motifs; Animals; Binding Sites; Electrophoresis, Polyacrylamide Gel; Humans; Lipid Bilayers; Liposomes; Magnetic Resonance Spectroscopy; Nanostructures; Phosphatidylcholines; Phosphatidylserines; Protein Binding; Rats; Recombinant Proteins; SNARE Proteins; Synaptosomal-Associated Protein 25; Syntaxin 1; Vesicle-Associated Membrane Protein 2

This paper describes a method to form giant liposomes in solutions of physiologic ionic strength, such as phosphate buffered saline (PBS) or 150 mM KCl. Formation of these cell-sized liposomes proceeded from hybrid films of partially dried agarose and lipids. Hydrating the films of agarose and lipids in aqueous salt solutions resulted in swelling and partial dissolution of the hybrid films and in concomitant rapid formation of giant liposomes in high yield. This method did not require the presence of an electric field or specialized lipids; it generated giant liposomes from pure phosphatidylcholine lipids or from lipid mixtures that contained cholesterol or negatively charged lipids. Hybrid films of agarose and lipids even enabled the formation of giant liposomes in PBS from lipid compositions that are typically problematic for liposome formation, such as pure phosphatidylserine, pure phosphatidylglycerol, and asolectin. This paper discusses biophysical aspects of the formation of giant liposomes from hybrid films of agarose and lipids in comparison to established methods and shows that gentle hydration of hybrid films of agarose and lipids is a simple, rapid, and reproducible procedure to generate giant liposomes of various lipid compositions in solutions of physiologic ionic strength without the need for specialized equipment.

Topics: Biophysical Phenomena; Buffers; Cholesterol; Liposomes; Osmolar Concentration; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Sepharose; Sodium Chloride; Spectrometry, Fluorescence

Magainin 2, a polycationic peptide, displays bactericidal and tumoricidal activity, presumably interacting with negatively charged phospholipids in the membrane hosts. In this work, we investigate the role played by the lipid head-group in the interactions and self-association of magainin 2 during pore formation in lipid bilayers. Two methods are used: single-channel and macroscopic incorporation into planar lipid membranes. Single-channel incorporation showed that magainin 2 did not interact with zwitterionic membranes, while the addition of negatively charged dioleoylphosphatidylglycerol to the membrane leads to channel formation. On the other hand, magainin 2 did not form channels in membranes made up of dioleoylphosphatidylserine (DOPS), although the addition of ergosterol to DOPS membranes leads to channel formation. This finding could indicate that ergosterol may be a possible target of magainin 2 in fungal membranes. Further support for this hypothesis comes from experiments in which the addition of ergosterol to palmitoyloleoylphosphatidylcholine membranes induced channel formation. Besides the role of negatively charged membranes, this study has shown that magainin 2 also forms channels in membranes lacking heads, such as monoolein and oxidized cholesterol, indicating an interaction of magainin 2 with acyl chains and cholesterol, respectively. This finding provides further evidence that peptide binding and assembly in lipid membranes is a complex process driven by electrostatic and/or hydrophobic interactions, depending on the structure of the peptide and the membrane composition.

Topics: Antimicrobial Cationic Peptides; Electric Conductivity; Electrochemistry; Ergosterol; Hydrophobic and Hydrophilic Interactions; Ion Channel Gating; Ion Channels; Lipid Bilayers; Lipids; Magainins; Membrane Potentials; Membranes, Artificial; Permeability; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylserines; Porosity; Static Electricity; Xenopus Proteins

Although neither the physiological function nor the mechanism of action of sterol carrier protein 2 (SCP(2)) is yet completely clear, it is thought that SCP(2) interacts with membranes to elicit its biological effects. The results presented here show that the SCP(2) N-terminus, composed of two amphipathic alpha-helices, interacted preferentially with highly curved but not lower-curvature membranes containing anionic phospholipid. CD spectra of SCP(2) showed up to 1. 2-fold increased alpha-helical content, on the interaction of SCP(2) with small unilamellar vesicles (SUV) (median radius 10-14 nm) but less with large unilamellar vesicles (LUV) (median radius 52-60 nm). Although enhanced interaction with the SUV membranes was due in part to the radius of curvature and to the greater exposure of acidic phospholipid in the outer leaflet of the bilayer, simply increasing the molar percentage of acidic phospholipid in the LUV membranes had much less effect on SCP(2) binding. A similar preferential interaction was observed with highly curved SUV as opposed to LUV for the SCP(2) N-terminal peptide (1-32)SCP(2) as well as structurally modified peptides in the order (1-32)SCP(2)=(10-32)SCP(2)>(1-24)SCP(2)>>(1-E20-32)SCP(2). The CD results were confirmed with an independent filtration binding assay, which showed that SCP(2) bound 5-fold more to SUV than LUV, whereas its N-terminal peptides bound up to 4-fold better in the order (1-32)SCP(2)=(10-32)SCP(2)>(1-24)SCP(2)>(1-E20-32)SCP(2). Finally, cholesterol potentiated the binding of SCP(2) and N-terminal peptides to anionic-phospholipid-containing SUV but not LUV. These findings were consistent with the SCP(2) N-terminus being a membrane-binding domain that was highly dependent on membrane surface curvature as well as on lipid composition.

Topics: Binding Sites; Carrier Proteins; Cholesterol; Circular Dichroism; Liposomes; Molecular Conformation; Particle Size; Peptide Fragments; Phosphatidylcholines; Phosphatidylserines; Plant Proteins; Protein Binding; Protein Structure, Secondary

The isothermal phase behavior of three gramicidin A'/phospholipid mixtures was investigated by an equilibrium Ca(2+)-binding technique. The phospholipid component was 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS), or POPS/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at a constant mole ratio of 1/4. The bulk aqueous free Ca2+ concentration, [Ca2+]*f, in equilibrium with one or two gramicidin A'/phospholipid fluid phases and a small amount of the Ca (phosphatidylserine)2 gel phase, was measured as a function of composition at 20 degrees C by use of chromophoric high-affinity Ca2+ chelators. The coexistence of two gramicidin A'/phospholipid fluid phases was detected by an invariance in [Ca2+]*f over the range of compositions throughout which the two phases coexist. The compositions of the two coexisting phases are determined by the compositions at which the invariance in [Ca2+]*f begins and ends. With each of the gramicidin A'/phospholipid mixtures, we estimate that the composition of the gramicidin-poor phase is 0.03-0.04 mole fraction gramicidin A' and the composition of the gramicidin-rich phase is 0.13-0.14 mole fraction gramicidin A'. Characterization of these phases by low-angle X-ray diffraction revealed that, in each case, the gramicidin-poor phase is an L alpha phase and the gramicidin-rich phase is an HII phase. The isothermal phase behavior of gramicidin A'/POPC mixtures at approximately 23 degrees C, as determined by low-angle X-ray diffraction, was found to be similar to that of the other gramicidin A'/phospholipid mixtures.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Binding Sites; Calcium; Chelating Agents; Gramicidin; Kinetics; Magnetic Resonance Spectroscopy; Phosphatidylcholines; Phosphatidylserines; Phosphorus Radioisotopes; Reference Standards; X-Ray Diffraction