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Neuronal fusion mediated by soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) is a fundamental cellular process by which two initially distinct membranes merge resulting in one interconnected structure to release neurotransmitters into the presynaptic cleft. To get access to the different stages of the fusion process, several in vitro assays have been developed. In this review, we provide a short overview of the current in vitro single vesicle fusion assays. Among those assays, we developed a single vesicle assay based on pore-spanning membranes (PSMs) on micrometre-sized pores in silicon, which might overcome some of the drawbacks associated with the other membrane architectures used for investigating fusion processes. Prepared by spreading of giant unilamellar vesicles with reconstituted t-SNAREs, PSMs provide an alternative tool to supported lipid bilayers to measure single vesicle fusion events by means of fluorescence microscopy. Here, we discuss the diffusive behaviour of the reconstituted membrane components as well as that of the fusing synthetic vesicles with reconstituted synaptobrevin 2 (v-SNARE). We compare our results with those obtained if the synthetic vesicles are replaced by natural chromaffin granules under otherwise identical conditions. The fusion efficiency as well as the different fusion states observable in this assay by means of both lipid mixing and content release are illuminated. [ABSTRACT FROM AUTHOR]

Gb3 glycosphingolipids are the specific receptors for bacterial Shiga toxin. Whereas the trisaccharidic head group of Gb3 defines the specificity of Shiga toxin binding, the lipophilic part composed of sphingosine and different fatty acids is suggested to determine its localization within membranes impacting membrane organisation and protein binding eventually leading to protein internalisation. While most studies use Gb3 extracts, chemical synthesis provides a unique tool to access different tailor-made Gb3 glycosphingolipids. In this review, strategies to synthesize these complex glycosphingolipids are presented. Special emphasis is put on the preparation of Gb3 molecules differing only in their fatty acid part (saturated, unsaturated, α-hydroxylated and both, unsaturated and α-hydroxylated). With these molecules in hand, it became possible to investigate the phase behaviour of liquid ordered/liquid disordered supported membranes doped with the Gb3 species by means of fluorescence and atomic force microscopy. The results clearly highlight the influence of the different fatty acids of the Gb3 sphingolipids on the phase behaviour and the binding properties of Shiga toxin B subunits, even though the membranes were only doped with 5 mol% of the receptor lipid. To obtain fluorescent Gb3 derivatives, either fatty acid labelled Gb3 molecules or head group labelled ones were synthesized. These molecules enabled us to address the question, where the Gb3 sphingolipids are localized prior protein binding by means of fluorescence microscopy on giant unilamellar vesicles. The results again demonstrate that the fatty acid of Gb3 plays a pivotal role for the overall membrane organisation. [ABSTRACT FROM AUTHOR]

Green propolis, a mixture of beeswax and resinous compounds processed by Apis mellifera, displays several pharmacological properties. Artepillin C, the major compound in green propolis, consists of two prenylated groups bound to a phenyl group. Several studies have focused on the therapeutic effects of Artepillin C, but there is no evidence that it interacts with amphiphilic aggregates to mimic cell membranes. We have experimentally and computationally examined the interaction between Artepillin C and model membranes composed of dimyristoylphosphatidylcholine (DMPC) because phosphatidylcholine (PC) is one of the most abundant phospholipids in eukaryotic cell membranes. PC is located in both outer and inner leaflets and has been used as a simplified membrane model and a non-specific target to study the action of amphiphilic molecules with therapeutic effects. Experimental results indicated that Artepillin C adsorbed onto the DMPC monolayers. Its presence in the lipid suspension pointed to an increased tendency toward unilamellar vesicles and to decreased bilayer thickness. Artepillin C caused point defects in the lipid structure, which eliminated the ripple phase and the pre-transition in thermotropic chain melting. According to molecular dynamics (MD) simulations, (1) Artepillin C aggregated in the aqueous phase before it entered the bilayer; (2) Artepillin C was oriented along the direction normal to the surface; (3) the negatively charged group on Artepillin C was accommodated in the polar region of the membrane; and (4) thinner regions emerged around the Artepillin C molecules. These results help an understanding of the molecular mechanisms underlying the biological action of propolis. [ABSTRACT FROM AUTHOR]

Static and time-resolved fluorescence of tryptophan and ortho-aminobenzoic acid was used to investigate the interaction of the synthetic antimicrobial peptide L1A (IDGLKAIWKKVADLLKNT-NH 2) with POPC and POPC:POPG. N-acetylated (Ac-L1A) and N-terminus covalently bonded ortho-aminobenzoic acid (Abz-L1A-W8V) were also used. Static fluorescence and quenching by acrylamide showed that the peptides adsorption to the lipid bilayers was accompanied by spectral blue shift and by a decrease in fluorescence quenching, indicating that the peptides moved to a less polar environment probably buried in the lipidic phase of the vesicles. These results also suggest that the loss of the N-terminus charge allowed deeper fluorophore insertion in the bilayer. Despite the local character of spectroscopic information, conclusions can be drawn about the peptides as a whole. The dynamic behaviors of the peptides are such that the mean intensity lifetimes, the long correlation time and the residual anisotropy at long times increased when the peptides adsorb in lipid vesicles, being larger in anionic vesicles. From the steady-state increase in fluorescence intensity and anisotropy, we observed that the partition coefficient of peptides L1A and its Abz analog in both types of vesicles are higher than the acetylated analog; moreover, the affinity to the anionic vesicle is higher than to the zwitterionic. [ABSTRACT FROM AUTHOR]

The interactions of the antimicrobial peptide maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH) with model phospholipid membranes were studied by use of dual polarisation interferometry and neutron reflectometry and dimyristoylphosphatidylcholine (DMPC) and mixed DMPC-dimyristoylphosphatidylglycerol (DMPG)-supported lipid bilayers chosen to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC bilayers concentration-dependent binding and increasing perturbation of bilayer order by maculatin were observed. By contrast, in mixed DMPC-DMPG bilayers, maculatin interacted more strongly and in a concentration-dependent manner with retention of bilayer lipid order and structure, consistent with pore formation. These results emphasise the importance of membrane charge in mediating antimicrobial peptide activity and emphasise the importance of using complementary methods of analysis in probing the mode of action of antimicrobial peptides. [ABSTRACT FROM AUTHOR]

Progress in the determination of structure and fluctuation spectrum of a floating bilayer system, as well as potential applications for biological studies, is reviewed. The system described here was first introduced by Charitat et al. (Eur Phys J B 8:583-593, ) and consists of a planar bilayer floating at 2-3 nm away from an adsorbed one on a solid surface in contact with bulk water. This model has been widely used for surface scattering studies using both neutrons and synchrotron radiation and its use in studies of relevance for physics and biology research areas will be described, together with the progress towards the production of complex biomimetic samples for use with scattering techniques. [ABSTRACT FROM AUTHOR]

Understanding biological processes assumes a detailed understanding of the interaction of all involved molecules. Here the effect of the peptide hormone angiotensin II (Ang II), an agonist of the angiotensin receptors, on the structure of unilamellar and multilamellar dimyristoyl phosphatidylcholine vesicles was studied by small angle neutron scattering, dynamic light scattering and differential scanning calorimetry. The calorimetry data indicate a weak interaction of Ang II with the surface of the membrane bilayer, as the pretransition persists during all experiments, and the main transition is only slightly shifted towards higher temperatures. From the SANS data we were able to confirm the calorimetric data and verify the interaction of the hormone with the membrane surface. At low temperatures, when the lipid molecules are in the gel phase, more precisely in the ripple phase, the peptide penetrates in the head group core, but due to the close packing of the acyl chains, the hydrophobic region is not affected. In a temperature region below but close to the region of the phase transition, the hydrophibic core starts to be affected by the peptide, and the same is true for the fluid phase. Upon binding of the peptide, the thickness of the head group increases, and the scattering length density of the head group starts to rise with increasing peptide concentrations. This interaction and binding to the membrane surface may be relevant for the relocation, binding and reconstitution of the angiotensin receptors into the membrane. Second, the peptide adsorption to the membrane surface may contribute to the binding of Ang II in the active site of the receptor. [ABSTRACT FROM AUTHOR]

The interaction of the synthetic antimicrobial peptide P5 (KWKKLLKKPLLKKLLKKL-NH) with model phospholipid membranes was studied using solid-state NMR and circular dichroism (CD) spectroscopy. P5 peptide had little secondary structure in buffer, but addition of large unilamellar vesicles (LUV) composed of dimyristoylphosphatidylcholine (DMPC) increased the β-sheet content to ~20%. Addition of negatively charged LUV, DMPC-dimyristoylphosphatidylglycerol (DMPG) 2:1, led to a substantial (~40%) increase of the α-helical conformation. The peptide structure did not change significantly above and below the phospholipid phase transition temperature. P5 peptide interacted differently with DMPC bilayers with deuterated acyl chains (d-DMPC) and mixed d-DMPC-DMPG bilayers, used to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC vesicles, P5 peptide had no significant interaction apart from slightly perturbing the upper region of the lipid acyl chain with minimum effect at the terminal methyl groups. By contrast, in the DMPC-DMPG vesicles the peptide increased disorder throughout the entire acyl chain of DMPC in the mixed bilayer. P5 promoted disordering of the headgroup of neutral membranes, observed by P NMR. However, no perturbations in the T relaxation nor the T- values were observed at 30°C, although a slight change in the dynamics of the headgroup at 20°C was noticeable compared with peptide-free vesicles. However, the P5 peptide caused similar perturbations of the headgroup of negatively charged vesicles at both temperatures. These data correlate with the non-haemolytic activity of the P5 peptide against red blood cells (neutral membranes) while inhibiting bacterial growth (negatively charged membranes). [ABSTRACT FROM AUTHOR]

Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus. It has been suggested that the N-terminal part, which contains a conserved intramolecular disulfide bond between residues 2 and 7, interacts with membranes, ultimately leading to membrane damage and β-cell death. Here, we used variants of the hIAPP1–19 fragment and model membranes of phosphatidylcholine and phosphatidylserine (7:3, molar ratio) to examine the role of this disulfide in membrane interactions. We found that the disulfide bond has a minor effect on membrane insertion properties and peptide conformational behavior, as studied by monolayer techniques, 2H NMR, ThT-fluorescence, membrane leakage, and CD spectroscopy. The results suggest that the disulfide bond does not play a significant role in hIAPP–membrane interactions. Hence, the fact that this bond is conserved is most likely related exclusively to the biological activity of IAPP as a hormone. [ABSTRACT FROM AUTHOR]

Langmuir monolayers of dioctadecyldimethyammonium bromide and its interaction with the natural mucopolysaccharide hyaluronic acid are studied using thermodynamic methods and X-ray diffraction at grazing incidence. The 2D crystalline lattice parameters of different phases are determined. The monolayer compressibility, the linear crystalline compressibility components and the thermoelastic expansion coefficient are evaluated. The biopolymer stabilises the monolayer structural properties, increases the collapse pressure and the correlation length of the 2D crystalline domains. The results show that this lipid has a potential for developing of stabilised drug delivery systems of anionic biopolymers like hyaluronic acid, oligomers and genes. [ABSTRACT FROM AUTHOR]

In this work thin liquid films (TLFs) and monolayers at the air/water interface formed by dimyristoylphosphatidylcholine (DMPC) and by DMPC mixed with poly ethylene glycols (PEGs) and dimyristoylphosphatidylethanolamine (DMPE) linked PEGs were studied. Film forming dispersions were composed of two types of particles: liposomes and micelles. TLFs stability, threshold concentration C t (i.e., the minimum one for stable film formation), and hydrodynamic behavior were measured. At equivalent conditions, DMPC films were Newton black films (real bilayers), while DMPE-PEGs films were much thicker with free water between the monolayers. DMPE-PEG addition to DMPC films caused both C t decrease (depending on PEG moiety length and Mw) and change of TLF formation mechanism. TLFs’ hydrodynamic behavior also strongly depended on DMPE-PEG content and Mw. It was observed that thinning of the DMPC and DMPE-PEGs films continued to different film types and thickness, being much thicker for the latter films. Addition of free PEGs (PEG-200/6000) did not alter TLF type or stability, but changed TLF thinning time, confirming that free PEGs with Mw<8000 could not penetrate in the membrane and alter “near-membrane” water layer viscosity. Monolayer studies showed improved formation kinetics of both adsorbed and spread films, decrease of surface tension (equilibrium and dynamic), and of film compression/decompression histeresis area in DMPE-PEGs monolayers compared with DMPC pure films. Our study shows that combining the models of phospholipid TLFs and monolayers provide the opportunity to investigate the properties of membrane surface and to clarify some mechanisms of its interactions with membrane-active agents. [ABSTRACT FROM AUTHOR]

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 compound’s antibacterial effects. [ABSTRACT FROM AUTHOR]

Interaction of the pentene antibiotic filipin with dimyristoylphosphatidylcholine (DMPC) membranes has been monitored by H-NMR, circular dichroism (CD), electronic absorption and fluorescence in the temperature range 10° to 60°C. Interaction appears to depend on whether filipin is added before or after membrane formation and also upon the temperature of the system. When filipin is added to preformed DMPC large unilamellar vesicles (LUV), the association constants, as determined by electronic absorption are 39×10 M, 15×10 M and 0.6×10 M at 15°, 30° and 50°C, respectively. Under identical conditions, CD spectra of bound filipin exhibit features characteristic of an aggregation over the whole temperature range. When filipin is incorporated in membranes during their preparation, the H-NMR spectra of deuterated DMPC indicate that the drug has a slight disordering effect on the lipid matrix below the temperature, T,of the gel-to-fluid phase transition and above T+11°C. Between these two temperature boundaries the system consists of two lipid regions of very different dynamic properties. One of the regions, which is attributed to a filipin-lipid complex, has the properties of gel-like lipids whereas the other has those of fluid-like lipids. The latter domain is however more ordered than the pure lipid at corresponding temperatures. CD spectra under the same conditions are found to be identical to spectra when the drug is added to preformed membranes, only in the region Tto T+11°C. Filipin induced carboxyfluorescein release from DMPC-LUV is found to be complete when the filipin-to-lipid ratio is near 1, for temperatures below T+11°C. Results are compared to previous data on amphotericin B and provide evidence that the gel-like structure of phospholipid and membrane permeation may be induced by filipin even in the absence of cholesterol. [ABSTRACT FROM AUTHOR]

A model is proposed for the effect of gramicidin A′ on the order and structure of phospholipid dispersions. According to this model, the addition of gramicidin A′ influences the surrounding lipids via two independent mechanisms. The first arises from a drop in surface pressure for those lipids substantially bounded by gramicidin A′. The second mechanism arises from the increase in the phospholipid headgroup spacing due to the small polar region of the polypeptide. The model provides an explanation for the currently available NMR, X-ray diffraction and Langmuir monolayer results. The model also suggests mechanisms for the ability of gramicidin A′ to trigger a transition of the lipid from the lamellar to hexagonal II phase, the dependence of this transition on the lipid chain length and the formation of a lamellar phase with lysophosphatidylcholine. [ABSTRACT FROM AUTHOR]

The translational diffusion of pyrene, pyrene butyric acid and pyrene decanoic acid has been determined in phosphatidylcholine bilayers of different chain length and under pressure up to 200 bars. In the liquid crystalline phase and at a given temperature the diffusion decreases with increasing chain length. At a constant reduced temperature, T (about 10 K above the transition temperature), long chain lipids exhibit the fastest diffusion which is in disagreement with hydrodynamic models but favours free volume models for diffusion in lipid bilayers. The volume of activation, V, calculated from the decrease of the diffusion coefficient with pressure, ∂ln D/∂ P, depends on lipid chain length. V decreases with decreasing lipid chain length at a given temperature, T=65°C, and increases at the reduced temperature. These results are again in agreement with the dependence of the diffusion on lipid chain length and therefore with the free volume model. [ABSTRACT FROM AUTHOR]

In phosphatidylserine membranes the decrease in the conductance of the gramicidin A single channel caused by calcium is attributed to a reduction of surface potential and to a direct blocking of the pore (Apell et al. 1979). The aim of this paper is to make a, quantitative evaluation of these two effects. We recorded the conductance of gramicidin single channels in 100 m M KCl in the presence of different amounts of CaCl, MgCl or TEACl. The ionic activities at the channel mouth were calculated using the Gouy-Chapman-Stern theory. Our experiments showed that even when the K activity at the channel mouth was estimated to be the same, the single channel conductance was lower if divalent cations were present. This effect is attributed to a blocking action of these ions. [ABSTRACT FROM AUTHOR]

Physical and biophysical mechanisms of mechano-sensitivity of cell membranes are reviewed. The possible roles of the lipid matrix and of the cytoskeleton in membrane mechanoreception are discussed. Techniques for generation of static strains and dynamic curvatures of membrane patches are considered. A unified model for stress-activated and stress-inactivated ion channels under static strains is described. A review of work on stress-sensitive pores in lipid-peptide model membranes is presented. The possible role of flexoelectricity in mechano-electric transduction, e.g. in auditory receptors is discussed. Studies of flexoelectricity in model lipid membranes, lipid-peptide membranes and natural membranes containing ion channels are reviewed. Finally, possible applications in molecular electronics of mechanosensors employing some of the recognized principles of mechano-electric transduction in natural membranes are discussed. [ABSTRACT FROM AUTHOR]

The action of the relaxing agent dantrolene on dipalmitoylphosphatidylcholine (DPPC) model membranes in the presence and absence of the general anesthetic halothane has been investigated by DSC and P-NMR. Dantrolene has a weak effect on both the thermodynamic and NMR parameters of the pure model membrane. When halothane is present in the system, the relaxing agent acts to counterbalance the strong anesthetic-induced membrane pertubation. This is reflected in DSC experiments by a change of the enthalpy variation (ΔH) and of the main gel-to-fluid phase transition temperature (T) towards the values of the pure lipid system. The amount of halothane-induced small tumbling vesicles, as detected by P-NMR by the superposition of an isotropic line on a lamellar-type powder spectrum, is considerably reduced upon dantrolene addition. This means that the relaxing agent 'cures' the membrane de-structuring action promoted by halothane. Membranes first treated with dantrolene are also protected from the halothane perturbation. So, the relaxing agent is both 'curative' and 'preventative' against halothane. The optimum effect is obtained for 1 dantrolene molecule per ca 34 halothane molecules. The mechanisms of action were discussed in relation to membrane fluidity. [ABSTRACT FROM AUTHOR]

The effect of the volatile anaesthetic halothane on the structure and dynamics of lipid multilayers (dimyristoyl- and dipalmitoylphosphatidylcholine, DM-and DP-PC, aqueous dispersions) was studied using Differential Scanning Calorimetry (DSC), Freeze Fracture Electron Microscopy and solid state phosphorus-31 Nuclear Magnetic Resonance (P-NMR). The action of the drug depends upon the halothane-to-lipid molar ratio, Ri, and temperature. With DPPC lipids, three main regions can be distinguished: i) 0 < Ri < 0.7, ii) 0.7 < Ri < 2 and iii) Ri > 2. As Ri increases in the first region, a linear decrease in the main gel-to-fluid phase transition temperature ( T, a broadening in the DSC transition peak and a lowering in the enthalpy variation (Δ H), are observed. A minimum in Δ H is reached at Ri=0.7. In this region, P-NMR spectra indicate that the multibilayer structure is maintained. In the second region, T still decreases with the same slope, but Δ H increases up to a plateau value for Ri=2. In the lipid fluid phase, an isotropic NMR line appears superimposed on the powder pattern that corresponds to a lamellar phase. For Ri > 2, T and Δ H remain almost constant. At values of temperature that are greater than T a growing isotropic line occurs in P-NMR spectra. This means a new supramolecular structure made of lipids and halothane is stabilized. This structure has been characterized as small vesicles of about 400 Å to 600 Å diameter by Freeze Fracture electron microscopy observations. With DMPC and low ratios ( Ri < 2), DSC and NMR results are similar to those obtained for DPPC. However, the minimum Δ H is reached at Ri=0.2 and the decrease in T is faster than for DPPC when Ri increases from 0. For Ri > 2, while T and Δ H remain constant as in the case of DPPC, P-NMR spectra of DMPC systems show a superimposition of an isotropic line and two powder patterns, which correspond to small tumbling vesicles, a possible hexagonal phase and a lamellar phase respectively. Halothane, thus acts on model membranes in two different steps: at low Ri the bilayer is disturbed but keeps its structure. Whereas for higher drug concentrations, a new organization of lipids seems to be stabilized for T > T. [ABSTRACT FROM AUTHOR]