Interaction of the eukaryotic pore-forming cytolysin equinatoxin II with model membranes: 19F NMR studies

Sea anemones produce a family of 18-20 kDa proteins, the actinoporins, which lyse cells by forming pores in cell membranes. Sphingomyelin plays an important role in their lytic activity, with membranes lacking this lipid being largely refractory to these toxins. As a means of characterising membrane binding by the actinoporin equinatoxin II (EqTII), we have used 19F NMR to probe the environment of Trp residues in the presence of micelles and bicelles. Trp was chosen as previous data from mutational studies and truncated analogues had identified the N-terminal helix of EqTII and the surface aromatic cluster including tryptophan residues 112 and 116 as being important for membrane interactions. The five tryptophan residues were replaced with 5-fluorotryptophan and assigned by site-directed mutagenesis. The 19F resonance of W112 was most affected in the presence of phospholipid micelles or bicelles, followed by W116, with further change induced by the addition of sphingomyelin. Although binding to phosphatidylcholine is not sufficient to enable pore formation in bilayer membranes, this interaction had a greater effect on the tryptophan residues in our studies than the subsequent interaction with sphingomyelin. Furthermore, sphingomyelin had a direct effect on EqTII in both model membranes, so its role in EqTII pore formation involves more than simply an indirect effect mediated via bulk lipid properties. The lack of change in chemical shift for W149 even in the presence of sphingomyelin indicates that, at least in the model membranes studied here, interaction with sphingomyelin was not sufficient to trigger dissociation of the N-terminal helix from the beta-sandwich, which forms the bulk of the protein.     

Interaction of ferredoxin:NADP oxidoreductase with model membranes

The ferredoxin:NADP⁺ oxidoreductase (FNR) is a plant enzyme, catalyzing the last step of photosynthetic linear electron transport, and involved also in cyclic electron transport around photosystem I. In this study we present the first evidence of FNR (isolated from spinach and from wheat) interaction directly with a model membrane without the mediation of any additional protein. The monomolecular layer technique measurements showed a significant increase in surface pressure after the injection of enzyme solution beneath a monolayer consisting of chloroplast lipids: monogalactosyldiacylglycerol or digalactosyldiacylglycerol. An ATR FTIR study revealed also the presence of FNR in a bilayer composed of these lipids. The secondary structure of the protein was significantly impaired by lipids, as with a pH-induced shift. The stabilization of FNR in the presence of lipids leads to an increase in the rate of NADPH-dependent reduction of dibromothymoquinone catalyzed by the enzyme. The biological significance of FNR-membrane interaction is discussed.     

Interaction of the cationic form of amphiphilic drugs with phosphatidylcholine model membranes. Competition with lanthanide ions

Model membranes (liposomes) of egg yolk phosphatidylcholine were exposed to the charged (cationic) form of amphiphilic drugs (procaine, tetracaine, metroprolol, alprenolol and propranolol). Drug analysis by ultraviolet light absorption of the bulk solution after centrifugation separation was used to determine the amount of drug bound to the membranes. Microelectrophoresis was employed to measure the change in the zeta-potential after drug adsorption. Binding constants were derived by simulating the experimental curves with a theoretical model which considers the electrostatic effects (Gouy-Chapman theory). Analogous experiments were carried out for the adsorption of Eu3+. Metal analysis was made by three different methods. Good agreement between the centrifugation and electrophoresis experiments was obtained for reasonable positions of the plane of shear relative to the positional plane of the bound ions. Displacement of Eu3+ from vesicles upon addition of drug cations was followed by 31P-NMR. The competition experiments were numerically simulated. The Eu3+ binding was assumed to obey a mass action type equilibrium, whereas the drug binding was described by a Henry's law partition. The binding constants for the drugs in the competition experiments followed the same order as in the absence of Eu3+. However, the numerical values had to be reduced. The effect of anions was studied.     

Fluorescence and ESR spectroscopy studies on the interaction of isoflavone genistein with biological and model membranes

Genistein (5,7,4′-trihydroxyisoflavone) the common soy beans isoflavone has attracted scientific interest due to its antioxidant, estrogenic, antiangiogenic and aniticancer activities. The aim of the present study was to investigate the interaction of genistein with biological (erythrocyte) and model membranes (dimyristoyl- and dipalmitoylphosphatidylcholine). Using Laurdan and Prodan as fluorescent probes, we demonstrated phase behavior and membrane fluidity changes induced by genistein. ESR spectroscopy revealed alterations caused by genistein in membrane domains structure and mobility of spin probes with free radicals located at different depths of membrane. The method of ESR spectra decomposition and computer simulation of the recorded spectra were used in order to visualize domain coexistence by GHOST condensation method. Fluorescence and ESR spectroscopy experiments performed at different temperatures enabled us to observe the effect of isoflavone on phospholipid bilayers in either gel or liquid crystalline phase. It was concluded that genistein preferentially intercalated into lipid headgroup region, to some extent into polar–apolar interface and only in minimal degree into hydrophobic core of the membrane. According to our best knowledge this is the first study on modification of domain structure of membranes by genistein.     

Studies of nonlinear electrical effects of model membranes

The structure of lipid bilayer membranes is investigated by analysis of the harmonics generated by electrical ac excitation with dc bias. For the detection of nonlinear effects this method is complementary to current relaxation experiments and may be suitable for gating current measurements at nerve membranes.Communication presented at the Biomembrane Symposium of the Deutsche Forschungsgemeinschaft, Heidelberg, October 1976     

Detailed molecular dynamics simulations of model biological membranes containing cholesterol

Detailed molecular dynamics simulations performed to study the nature of lipid raft domains that appear in model membranes are reviewed in this paper. The described simulations were performed on hydrated bilayers containing binary mixtures of cholesterol with phospholipids and also on ternary mixtures containing cholesterol, a phospholipid with a high main transition temperature T_m, and a phospholipid with a low transition temperature T_m. These simulations provide qualitative and semi-quantitative information about cholesterol-lipid interactions and also a testing ground for major assumptions made to explain the nature of lipid rafts in model membranes.     

Towards high-resolution H-NMR in biological membranes: magic angle spinning of bicelles

Proton line narrowing in biomembranes spun at the magic angle, for spinning speeds greater than 7 kHz, was investigated in two ways: increasing the field strength from 200 to 800 MHz and changing the membrane fluidity. The resolution that one can obtain on natural lipid membranes under the form of liposomes is 0.019 ppm at 800 MHz. On the other hand, spinning bicelles (disk-like model membranes made of synthetic long and short chain lipids) at the magic angle decreases the line width by an additional factor of 3 provided the bicelle is subjected to large orientational disorder. This leads to proton line widths of the order of 6 Hz at 500 MHz. The conjunction of high field, magic angle spinning and use of bicelle membranes should prove to be useful to solve membrane protein structure in a membrane environment.     

Location and orientation of Triclosan in phospholipid model membranes

Triclosan is a hydrophobic antibacterial agent used in dermatological preparations and oral hygiene products. Although the molecular mechanism of action of this molecule has been attributed to inhibition of fatty acid biosynthesis, earlier work in our laboratories strongly suggested that the antibacterial action of Triclosan is mediated at least partly through its membranotropic effects. In order to assess its location in phospholipid membranes, high-resolution magic-angle spinning natural abundance ¹³C NMR of Triclosan embedded within egg yolk lecithin model membranes has been used to obtain ¹³C spin–lattice relaxation times for both Triclosan and lecithin carbon atoms in the presence of Gd³⁺ ions. The results indicate that Triclosan is localized in the upper region of the phospholipid membrane, its hydroxyl group residing in the vicinity of the C=O/C2 carbon atoms of the acyl chain of the phospholipid, and the rest of the Triclosan molecule is probably aligned in a nearly perpendicular orientation with respect to the phospholipid molecule. Intercalation of Triclosan into bacterial cell membranes likely compromises the functional integrity of those membranes, thereby accounting for at least some of this compounds antibacterial effects.Abbreviations COLOC correlation by long-range coupling - EYL egg yolk lecithin - HETCOR heteronuclear chemical-shift correlation - MAS magic-angle spinning - MLV multilamellar vesicles     

Specific interactions of sticholysin I with model membranes: An NMR study

Sticholysin I (StnI) is an actinoporin produced by the sea anemone Stichodactyla helianthus that binds biological and model membranes forming oligomeric pores. Both a surface cluster of aromatic rings and the N‐terminal region are involved in pore formation. To characterize the membrane binding by StnI, we have studied by ¹H‐NMR the environment of these regions in water and in the presence of membrane‐mimicking micelles. Unlike other peptides from homologous actinoporins, the synthetic peptide corresponding to residues 1–30 tends to form helix in water and is more helical in either trifluoroethanol or dodecylphosphocholine (DPC) micelles. In these environments, it forms a helix‐turn‐helix motif with the last α‐helical segment matching the native helix‐α₁ (residues 14–24) present in the complete protein. The first helix (residues 4–9) is less populated and is not present in the water‐soluble protein structure. The characterization of wild‐type StnI structure in micelles shows that the helix‐α₁ is maintained in its native structure and that this micellar environment does not provoke its detachment from the protein core. Finally, the study of the aromatic resonances has shown that the motional flexibility of specific rings is perturbed in the presence of micelles. On these bases, the implication of the aromatic rings of Trp‐111, Tyr‐112, Trp‐115, Tyr‐132, Tyr‐136, and Tyr‐137, in the interaction between StnI and the micelle is discussed. Based on all the findings, a revised model for StnI interaction with membranes is proposed, which accounts for differences in its behavior as compared with other highly homologous sticholysins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Probing molecular level interaction of oseltamivir with H5N1-NA and model membranes by molecular docking, multinuclear NMR and DSC methods

Structure-based drug design has led to the introduction of three drugs — oseltamivir (GS-4104), zanamivir (GG-167) and peramivir (RWJ-270201) which target the enzyme neuraminidase, for treatment of influenza infections. Using comparative docking studies we propose that more potent molecules against neuraminidase can be obtained by appending extra positively charged substituents at the C5 position of the oseltamivir skeleton. This provides an additional interaction with the enzyme and may overcome the problem of resistance encountered with these drugs. To get an insight into the transport and absorption of oseltamivir — the ethyl ester prodrug (GS-4104) as well as its mechanism of action, we have carried out ¹H, ¹³C, ³¹P NMR, DSC and TEM studies on GS-4104 with model membranes prepared from DMPC/DPPC/POPC. These studies reveal that interactions between GS-4104 and the membrane are both electrostatic (involving H-bonding) and hydrophobic (involving the hydrophobic chain and cyclohexene ring of GS-4104) in nature. The prodrug is seen to increase the fluidity as well as stabilize the bilayer phase of the membrane. This property may be responsible for preventing viral entry into the cells by preventing fusion of the virus outer coat with the cell membrane.     

Anomaly of transport of dipicrylamine anions across the central barrier of planar dipalmitoylphosphatidylcholine bilayer membranes at the phase transition

The rate of translocation of the hydrophobic ion dipicrylamine across planar lipid membranes formed from dipalmitoyllecithin in n-decane was determined by voltage jump relaxation experiments. The activation energy of the rate constant shows a change from a positive to a negative value at about 42°C near the main phase transition temperature of this lipid. Below this temperature, the rate constant was found to increase with decreasing temperature. This anomalous behaviour extends over a temperature range of at least 10 K and may be formally interpreted as an enhanced mobility of dipicrylamine in the solid state of the membrane.     

The influence of NBD fluorescent probe on model membranes containing POPC and DPPC

To investigate the effect of fluorescent probe on the properties of membranes, we studied model membranes composed of 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl 2-oleoyl-sn-glycero-3-phosphocholine (POPC) in the presence and absence of fluorescent probe. The morphology of giant unilamellar vesicles (GUVs) has been observed as a function of temperature and composition by fluorescence microscopy using NBD-DOPE or C₆-NBD-PC as the probe. The phase behavior of model membranes containing no fluorescent probe was investigated by ²H-NMR spectroscopy. We found that the bright phase observed on GUVs was the fluid phase enriched in POPC and the dark phase was the gel phase enriched in DPPC. NBD-DOPE and C₆-NBD-PC preferentially participated in the fluid-phase domains when GUVs were in the gel?+?fluid phase coexistence. Inclusion of both fluorescent probes (1?mol%) lowered the transition temperature of POPC/DPPC membranes. In addition, C₆-NBD-PC exhibited a stronger effect than NBD-DOPE, which was considered to be associated with the structures of fluorescent molecules.     

Farnesylated peptides in model membranes: a biophysical investigation

Protein prenylation plays an important role in signal transduction, protein-protein interactions, and the localization and association of proteins with membranes. Using three different techniques, this study physically characterizes the interactions between model dimyristoylphosphatidylcholine membranes and a series of farnesylated peptides. Magic angle spinning nuclear Overhauser enhancement spectroscopy and differential scanning calorimetry reveal that both charged [Ac-Asn-Lys-Asn-Cys-(farnesyl)-OMe and Ac-Asn-Lys-Asn-Cys-(farnesyl)-NH(2)] and uncharged [Ac-Cys-(farnesyl)-OMe and farnesol] species partition into dimyristoylphosphatidylcholine bilayers. Calorimetry and vesicle fluctuation analysis of giant unilamellar vesicles show that the charged peptides modestly decrease the main gel-fluid phase transition and markedly increase the bending rigidity of large unilamellar vesicles. Uncharged species, on the other hand, dramatically decrease the main phase transition and modestly decrease the bending rigidity. No difference with carboxyl methylation is detected.     

Interaction of peptides spanning the transmembrane domain of caveolin‐1 with model membranes

Caveolin‐1 has an atypical membrane‐spanning domain comprising of 34 residues. Caveolin‐1 targets to lipid droplets under certain conditions, where they are involved in signaling and cholesterol balance. In the present study, membrane association of synthetic peptides corresponding to the membrane‐spanning domain of caveolin‐1 has been investigated to obtain an insight into the topology of transmembrane region in the lipid bilayer and the effect of truncations in this sequence, as observed in the targeting to lipid droplets, by using model membranes. Fluorescence studies revealed strong association of the peptide corresponding to the membrane‐spanning domain of caveolin‐1 with anionic lipids as compared with zwitterionic lipids, which is consistent with the location of this domain in the cytoplasmic side of the plasma membrane. Association of a short 9 residue peptide corresponding to the C‐terminus of caveolin‐1 membrane‐spanning domain with lipid vesicles revealed the importance of this region for association with model membranes. Our investigations indicate that the peptide corresponding to the membrane‐spanning domain of caveolin‐1 does not span the lipid bilayer. We propose that both caveolin scaffolding domain and transmembrane segment of caveolin‐1 contribute to the strong association with the plasma membrane rendering the protein highly detergent resistant. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Interaction of antiaggregant molecule ajoene with membranes

The structure of ajoene, a molecule extracted from garlic, has been studied by ¹H-NMR and its interaction with model membranes by ¹H-, ²H-, ³¹-P-NMR and ESR experiments. This study clearly shows that the ajoene molecule is located deep in the layer and is close to the interlayer medium. Moreover while NMR experiments show that the membrane structure is only slightly affected by the presence of ajoene, ESR experiments reveal significant modifications in phospholipid dynamics. This interaction, observed before with the phenothiazine derivative, promazine, results in an increase of the membrane fluidity in its hydrophobic part and could be related to clinical properties of ajoene.     

NMR studies of drug interaction with membranes and membrane-associated proteins

This review focuses on the recent developments in the study of drug interactions with biological membranes and membrane-associated proteins using nuclear magnetic resonance (NMR) spectroscopy and other spectroscopic techniques. Emphasis is placed on a class of low-affinity neurological agents as exemplified by volatile general anesthetics and structurally related compounds. The technical aspects are reviewed of how to prepare membrane-mimetic systems and of NMR approaches that are either in current use or opening new prospects. A brief literature survey covers studies ranging from drug distribution in simplified lipid matrix to specific drug interaction with neuronal receptors reconstituted in complicated synthetic membrane systems.     

The influence of fatty acids on model cholesterol/phospholipid membranes

The aim of this work was to verify the influence of the saturated (SFA) (stearic acid) and the unsaturated (UFA) (oleic and alpha-linolenic) fatty acids on model cholesterol/phospholipid membranes. The experiments were based on the Langmuir monolayer technique. Cholesterol and phospholipid were mixed in the molar ratio that corresponds to the proportion of these lipids in the majority of natural human membranes. Into the binary cholesterol/phospholipid monolayers, various amounts of fatty acids were incorporated. Our investigations were based on the analysis of the interactions between molecules in ternary (cholesterol/phospholipids/fatty acid) mixtures, however, also binary (cholesterol/fatty acid and phospholipids/fatty acid) mixed system were examined. It was concluded that the influence of the fatty acids on model cholesterol/phospholipid membrane is closely connected with the shape of the fatty acid molecule, resulting from the saturation degree of the hydrocarbon chain. It was found that the saturated fatty acid makes the model membrane more rigid, while the presence of unsaturated fatty acid increases its fluidity. The increasing amount of stearic acid gradually destabilizes model membrane, however, this effect is the weakest at low content of SFA in the mixed monolayer. Unsaturated fatty acids in a small proportion make the membrane thermodynamically more stable, while higher content of UFA decreases membrane stability. This explains low proportion of the free fatty acids to other lipids in natural membrane.     

Characterization of the phase behavior of phosphonolipids in model and biological membranes by 31P-NMR

³¹P-NMR is used to characterize the phase behavior of phosphonolipids in both model and biological membranes. ($\text{1′,2′-}\text{Dipalmitoyl}\text{-sn-}\text{glyceryl)-2-aminoethylphosphonate}$ gives rise to static chemical shift tensor elements (?87, 5 and 63 ppm) which differ considerably from those reported for the analogous phospholipid, $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero-3-phosphoethanolamine}$ (?81, ?20 and 105 ppm). Phosphonolipid, as well as a mixture of phosphonolipid and $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero-3-phosphocholine}$, in aqueous dispersion gives rise to ³¹P spectra which may be interpreted in terms of lamellar structures. A mixture of phosphonolipid and egg phosphatidylethanolamine exhibits a bilayer-to-hexagonal phase transition with a concomitant decrease by one-half in the value of the ³¹P chemical shift anisotropies of both the phosphonate and phosphate resonances. The chemical shift anisotropy associated with phosphonolipid has been found to be consistently smaller than that observed for the analogous phospholipid. ³¹P-NMR spectra of total lipid extracts of Tetrahymena sp. indicate that both phospho- and phosphonolipids have a bilayer organization between ?20 and 20°C.     

Solution NMR studies of peptide-lipid interactions in model membranes

Abstract

Many important processes in life take place in or around the cell membranes. Lipids have different properties regarding their membrane-forming capacities, their mobility, shape, size and surface charge, and all of these factors influence the way that proteins and peptides interact with the membrane. In order for us to correctly understand these interactions, we need to be able to study all aspects of the interplay between lipids and peptides and proteins. Solution-state NMR offers a somewhat unique possibility to investigate structure, dynamics and location of proteins and peptides in bilayers. This review focuses on solution NMR as a tool for investigating peptide-lipid interaction, and special attention is given to the various membrane mimetics that are used to model the membrane. Examples from the field of cell-penetrating peptides and their lipid interactions will be given. The importance of studying lipid and peptide dynamics, which reflect on the effect that peptides have on bilayers, is highlighted, and in this respect, also the need for realistic membrane models.     

Biophysical studies of the interactions between 14-mer and 21-mer model amphipathic peptides and membranes: Insights on their modes of action

We have investigated the interactions between synthetic amphipathic peptides and zwitterionic model membranes. Peptides with 14 and 21 amino acids composed of leucines and phenylalanines modified by the addition of crown ethers have been synthesized. The 14-mer and 21-mer peptides both possess a helical amphipathic structure as revealed by circular dichroism. To shed light on their mechanism of membrane interaction, different complementary biophysical techniques have been used such as circular dichroism, fluorescence, membrane conductivity measurement and NMR spectroscopy. Results obtained by these different techniques show that the 14-mer peptide is a membrane perturbator that facilitate the leakage of species such as calcein and Na ions, while the 21-mer peptide acts as an ion channel. ³¹P solid-state NMR experiments on multilamellar vesicles reveal that the dynamics and/or orientation of the polar headgroups are greatly affected by the presence of the peptides. Similar results have also been obtained in mechanically oriented DLPC and DMPC bilayers where different acyl chain lengths seem to play a role in the interaction. On the other hand, ²H NMR experiments on multilamellar vesicles demonstrate that the acyl chain order is affected differently by the two peptides. Based on these studies, mechanisms of action are proposed for the 14-mer and 21-mer peptides with zwitterionic membranes.