Lipid-dependent pore formation by antimicrobial peptides arenicin-2 and melittin demonstrated by their proton transfer activity.

This work presents a comparative study of proton transfer activity (PTA) of two cationic (+6) antimicrobial peptides, β-structural arenicin-2 and α-helical melittin. A new approach was proposed for the detection of passive proton transfer by using proteoliposomes containing bacteriorhodopsin, which creates a small light-induced electrochemical proton gradient ∆ΔpH. Addition of several nanomoles of the peptides lowers ∆ΔpH that is proximately indicative of the pore formation.

Conformation of gramicidin A in Triton X-100 micelles from CD and FTIR data: a clean example of antiparallel double β5.6 helix formation.

The linear peptide gramicidin A (gA) forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization and dynamics of membrane channels. This polymorphic peptide can adopt two different types of structures, the helical dimer β6.3 ('channel state') and the double helical structure with two intertwined monomers.

Molecular mechanism of action of β-hairpin antimicrobial peptide arenicin: oligomeric structure in dodecylphosphocholine micelles and pore formation in planar lipid bilayers.

The membrane-active, cationic, β-hairpin peptide, arenicin, isolated from marine polychaeta Arenicola marina exhibits a broad spectrum of antimicrobial activity. The peptide in aqueous solution adopts the significantly twisted β-hairpin conformation without pronounced amphipathicity. To assess the mechanism of arenicin action, the spatial structure and backbone dynamics of the peptide in membrane-mimicking media and its pore-forming activity in planar lipid bilayers were studied.

[ESR study of the interaction of cytotoxin II with model membranes].

Cytotoxin II from the venom of the Central-Asian cobra Naja oxiana spin-labeled at Lys35 (SLCT II) was studied by ESR spectroscopy in aqueous solution and upon interaction with phospholipid vesicles from egg phosphatidylcholine or its mixture with dimyristoylphosphatidylglycerol (molar ratio 9:1). The distribution of SLCT II between the aqueous and lipid phases depended on the toxin and lipid concentrations and on the solution ionic strength. It was analyzed using the modified Gouy-Chapman equation that takes into account different charges of the cytotoxin in solution and in membrane.

[Synthesis and properties of fluorescent labeled detergents analogues of glycocholic acid].

For studying membrane processes with participation of detergents, fluorescent analogues of glycocholic acid containing p-hydroxybenzyl, 7-nitrobenz-2-oxa-1,3-diazol-4-yl, or fluorescein-5-thiocarbamoyl fluorophore in the glycyl moiety attached to glycocholic acid were synthesized. The fluorophores are in the probes near their carboxyl groups and, in membrane systems, should therefore be situated on the interface and be sensitive to phase transitions. The critical micelle concentrations were determined for the analogues and found to be close to those of cholate and glycocholate in the case of the first two compounds.

Synergetics of the Membrane Self-Assembly: A Micelle-to-Vesicle Transition.

A model approach is developed to study intermediate steps and transientstructures in a course of the membrane self-assembly. The approach isbased on investigation of mixed lipid/protein-detergent systems capable ofthe temperature induced transformation from a solubilized micellar stateto closed membrane vesicles. We performed a theoretical analysis ofself-assembling molecular structures formed in binary mixtures ofdimyristoylphosphatidylcholine (DMPC) and sodium cholate (NaC).

Structural polymorphism of gramicidin A channels: ion conductivity and spectral studies.

The relation between the various spatial structures of the gramicidin A channels and their ionic conductance has been studied. For this aim, various conformations of the peptide were pre-formed in liposomal bilayer and after subsequent fusion of liposomes with planar lipid bilayer the measured channel conductance was correlated with gramicidin structures established in liposomes. To form the single-stranded pi 6.

Temperature-induced micellar-lamellar transformation in binary mixtures of saturated phosphatidylcholines with sodium cholate.

The transition states of binary mixtures of dipalmitoyl- and dimyristoylphosphatidylcholines with sodium cholate at the reversible temperature-induced micellar-lamellar transformation were characterized by turbidimetry, electron microscopy, 31P NMR and differential scanning calorimetry. This transformation is triggered by the phospholipid acyl chain melting, and appears to include two structural pathways: (i) from discoidal mixed micelles to network-like structures composed of long interlaced rod-like micelles, then to multilayer membrane structures, and finally to multilamellar vesicles; and (ii) from discoidal micelles to membrane fragments and finally to unilamellar vesicles.

The double pi pi 5.6 helix of gramicidin A predominates in unsaturated lipid membranes.

The structure of the channel-forming polypeptide gramicidin A (GA) incorporated into phosphatidyl-choline (PC) liposomes has been studied as a function of the degree of unsaturation of the acyl chains of PC. The initial conformational state of GA in reconstituted bilayers is determined by the solvent in which the peptide and the lipid are initially co-dissolved, whereas the equilibrium conformational state (after heat incubation) is affected by the lipid structure rather than by the nature of the solvent. The conformational equilibrium of GA has been studied in liposomes prepared from PC having a variable number of double bonds in the fatty acid moiety, by circular dichroism and Fourier transform infrared.

The divalent cation-binding sites of gramicidin A transmembrane ion-channel.

The conductance of the gramicidin A single channels in glycerolmonooleate membranes is strongly reduced in the presence of Mn2+ cations. The nmr experiments were performed for N-terminal to N-terminal gramicidin A dimer formed by two right-handed single-stranded helixes incorporated into the sodium dodecyl sulfate micelles in the presence of Mn2+ ions. Dependence of the nonselective spin-lattice relaxation rates of the gramicidin A protons on Mn2+ concentration was analyzed to determine coordinates of the divalent cation binding sites.

Phospholipid topology and flip-flop in intestinal brush-border membrane.

The topological distribution of the two major phospholipids of brush-border membrane, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), has been investigated using brush-border membrane vesicles from rabbit small intestine. Bee venom phospholipase A2 and phosphatidylcholine exchange protein from bovine liver were used as membrane probes. It is shown that the brush-border membrane retains its integrity under conditions of phospholipase hydrolysis and intermembrane phospholipid exchange.

[Rapid transmembrane migration of phosphatidylcholine under the influence of cytochrome P-450].

The phosphatidylcholine exchange protein and bee venom phospholipase A2 were used to estimate the phosphatidylcholine accessibility in proteoliposome-containing cytochrome P-450. Without cytochrome P-450 phosphatidylcholine was only partly accessible for the both membrane probes. Incorporation of cytochrome P-450 into the liposomes resulted in a complete accessibility of phosphatidylcholine for the phosphatidylcholine exchange protein and phospholipase A2.

[Transmembrane migration of phosphatidylcholine and peroxidation of lipids in rat liver microsomes].

The exchangeability of phosphatidylcholine in microsomes differing in the degree of lipid peroxidation was determined by incubation of microsomes labelled in vivo with [methyl-14C]choline chloride with an excess of phosphatidylcholine liposomes and phosphatidyl-exchange protein from beef liver. The phosphatidylcholine peroxidation products were shown to act as substrates for the phosphatidylcholine-exchange protein. The level of exchangeable microsomal phosphatidylcholine did not depend on the degree of lipid peroxidation in the microsomes.

A sphingomyelin transfer protein in rat tumors and fetal liver.

The binding of the disaccharides methyl beta-D-lactoside and 2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-beta-D-galactopyranose [beta-D-Gal-(l leads to 3)-D-GalNAc] to peanut agglutinin was studied by ultraviolet difference spectroscopy. The magnitude of the difference spectra varied with the concentration of the carbohydrates; association constants and thermodynamic parameters were determined from titration experiments at different temperatures. The enthalpy and entropy changes for binding of methyl beta-D-lactoside were found to be delta H degree = -65 +/- 4 kJ mol-1, delta S degree = -156 +/- 14 J mol-1 K-1.

Investigation of the inside-outside distribution, intermembrane exchange and transbilayer movement of phospholipids in sonicated vesicles by shift reagent NMR.

1. A new NMR approach is described for the investigation of transbilayer asymmetry in phospholipid vesicles consisting of phosphatidylcholine and negatively charged phospholipids. The method makes use of the dependence of the psuedocontact shift of the N-methyl proton resonance induced by paramagnetic ions on the surface concentration of negatively charged phospholipids.

Affinity chromatography of the phosphatidylcholine exchange protein from bovine liver.

Affinity chromatography has been used to purify the phosphatidylcholine exchange protein from bovine liver. The affinity resin consisted of 1-acyl-2-(9-carboxy)nonyl-glycero-3-phosphocholine linked to AH-Sepharose 4 B via the carboxyl group. Application of a crude exchange protein fraction to the affinity column resulted in a complete adsorption of the phosphatidylcholine exchange protein.

Manipulation of phospholipid composition of membranes with the aid of lipid exchange proteins. Incorporation of phosphatidylcholine into protoplasts of Micrococcus lysodeikticus.

Incubation of Micrococcus lysodeikticus protoplasts with phosphatidylcholine liposomes and rat liver exchange proteins (pH 5.1 supernatant fraction) resulted in replacement of about one half of the bacterial total phospholipids by phosphatidylcholine. Protoplasts modified by phosphatidylcholine showed a decreased rate of oxidation of exogenous substrates (NADH, malate) and decreased ferricyanide reductase activity as compared to the initial protoplasts.