Fluorescence study of the fluidity and cooperativity of the phase transitions of zwitterionic and anionic liposomes confined in sol-gel glasses

The current work makes use of different fluorescent reporter molecules and fluorescent spectroscopic techniques to characterize the thermotropic, physical, and dynamical properties of large unilamellar liposomes formed from either 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-glycerol] (DMPG) encapsulated in sol-gel matrixes. In particular, cooperativity of the phase transition is analyzed from steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), the interfacial properties are studied by measuring the spectral shift of Laurdan, and the structural organization (heterogeneity) of the lipid bilayer is determined from the fluorescence lifetime of trans-parinaric acid (t-PnA). In addition, information regarding order and dynamical properties in the bulk hydrophobic core is obtained from time-resolved fluorescence anisotropy of t-PnA and 3-(4-(6-phenyl)-1,3,5-hexatrienyl)-phenylpropionic acid (PA-DPH). The spectroscopic study reveals that upon encapsulation, the basic thermodynamic properties as well as the fluidity of the lipid bilayer practically remain intact for DMPG liposomes but not for DMPC liposomes, whose lipid bilayer exhibits large gel-fluid heterogeneity. On the basis of these experimental results, electrostatic interactions between phospholipid polar heads and the porous surface of the host matrix seem to play a capital role for the preservation of the structural integrity of encapsulated bilayer.     

Interactions in lipid stabilised foam films

The interaction between lipid bilayers in water has been intensively studied over the last decades. Osmotic stress was applied to evaluate the forces between two approaching lipid bilayers in aqueous solution. The force–distance relation between lipid mono- or bilayers deposited on mica sheets using a surface force apparatus (SFA) was also measured. Lipid stabilised foam films offer another possibility to study the interactions between lipid monolayers. These films can be prepared comparatively easy with very good reproducibility. Foam films consist usually of two adsorbed surfactant monolayers separated by a layer of the aqueous solution from which the film is created. Their thickness can be conveniently measured using microinterferometric techniques. Studies with foam films deliver valuable information on the interactions between lipid membranes and especially their stability and permeability. Presenting inverse black lipid membrane (BLM) foam films supply information about the properties of the lipid self-organisation in bilayers. The present paper summarises results on microscopic lipid stabilised foam films by measuring their thickness and contact angle. Most of the presented results concern foam films prepared from dispersions of the zwitterionic lipid 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) and some of its mixtures with the anionic lipid — 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG).     

Effects of synthetic amphiphilic alpha-helical peptides on the electrochemical and structural properties of supported hybrid bilayers on gold

Amphiphilic alpha-helices were formed from designed synthetic peptides comprising alanine, phenylalanine, and lysine residues. The insertion of the alpha-helical peptides into hybrid bilayers assembled on gold was studied by a variety of methods to assess the resulting structural characteristics, such as electrical resistance and molecular orientation. Self-assembled monolayers (SAMs) of dodecanethiol (DDT); octadecanethiol (ODT); and 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) were formed on gold substrates with and without incorporated peptide. Supported hybrid bilayers and multilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were formed on SAMs by the "paint-freeze" method of bilayer formation. Modeling of electrochemical impedance spectroscopy data using equivalent electrochemical circuits revealed that the addition of peptide decreased dramatically the resistive element of the bilayer films while maintaining the value of the capacitive element, indicating successful incorporation of peptide into a well-formed bilayer. Near-edge X-ray absorption fine structure spectroscopy data provided evidence that the molecules in the SAMs and hybrid multilayers were ordered even in the presence of peptide. The peptide insertion into the SAM was confirmed by observing the pi* resonance peak correlating with phenylalanine and a peak in the nitrogen K-edge regime attributable to the peptide bond.     

Interaction and lipid-induced conformation of two cecropin-melittin hybrid peptides depend on peptide and membrane composition

The interaction of two hybrid peptides of cecropin A and melittin [CA(1-8)M(1-18) and CA(1-7)M(2-9)] with liposomes was studied by differential scanning calorimetry (DSC), circular dichroism (CD), and quasi-elastic light scattering (QELS). The study was carried out with large unilamellar vesicles (LUVs) of three different lipid compositions: 1,2-dimyristoil-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DMPG) and a binary mixture of DMPC/DMPG, in a wide range of peptide-to-lipid (P:L) molar ratios (0 to 1:7). DSC results indicate that, for both peptides, the interaction depends on membrane composition, with very different behavior for zwitterionic and anionic membranes. CD data show that, although the two peptides have different secondary structures in buffer (random coil for CA(1-7)M(2-9) and predominantly beta-sheet for CA(1-8)M(1-18)), they both adopt an alpha-helical structure in the presence of the membranes. Overall, results are compatible with a model involving a strong electrostatic surface interaction between the peptides and the negatively charged liposomes, which gives place to aggregation in the gel phase and precipitation after a threshold peptide concentration. In the case of zwitterionic membranes, a progressive surface coverage with peptide molecules destabilizes the membrane, eventually leading to membrane disruption. Moreover, delicate modulations in behavior were observed depending on the peptide.     

Structure and dynamics of phospholipid bilayers using recently developed general all-atom force fields

Two fully hydrated pure-species phospholipids bilayers, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), in the fluid phase and explicit solvent have been studied using molecular dynamics simulation. Atom interactions were modeled using recently developed force fields based on AMBER with full atomistic details. Several representative liquid phase properties for the structure and dynamics of lipids with different length of hydrocarbon chains and different level of saturation have been reproduced without artificially biasing the system in order to match experimental data. In particular, as the new GAFF (General Amber Force Field) has not been explicitly developed to reproduce lipid characteristics and is naturally compatible with standard AMBER nucleic acids and proteins parameters, it is here proven a promising tool to study mixed lipid-protein processes as protein activity dependence on membrane composition, permeation of solute across membranes, and other cellular processes.     

Conjugated polyelectrolyte supported bead based assays for phospholipase A2 activity

A fluorescence based assay for human serum-derived phospholipase activity has been developed in which cationic conjugated polyelectrolytes are supported on silica microspheres. The polymer-coated beads are overcoated with an anionic phospholipid (1,2-dimyristoyl-sn-glycero-3-[phospho- rac-(1-glycerol)) (DMPG) to provide "lipobeads" that serve as a sensor for PLA2. The lipid serves a dual role as a substrate for PLA2 and an agent to attenuate quenching of the polymer fluorescence by the external electron transfer quencher 9,10-anthraquinone-2,6-disulfonic acid (AQS). In this case quenching of the polymer fluorescence by AQS increases as the PLA2 digests the lipid. The lipid can also be used itself as a quencher and substrate by employing a small amount of energy transfer quencher substituted lipid in the DMPG. In this case the fluorescence of the polymer is quenched when the lipid layer is intact; as the enzyme digests the lipid, the fluorescence of the polymer is restored. The sensing of PLA2 activity has been studied both by monitoring fluorescence changes in a multiwell plate reader and by flow cytometry. The assay exhibits good sensitivity with EC50 values in the nanomolar range.     

Ferrocenylalkylthiolate labeling of defects in alkylthiol self-assembled monolayers on gold

Coverage defects in alkylthiol self-assembled monolayers (SAMs) are critically important to function related to electron transfer from soluble redox probes. There is therefore a need for an accurate and direct measurement of the number and type of coverage defect in a range of SAMs. Ferrocenyldodecanethiol (FcC(12)SH) has been assessed as an electrochemically-addressable label of coverage defects. It is shown that short time exposure of a SAM to FcC(12)SH leads to a quantifiable Fc coverage (Gamma(Fc)), with Gamma(Fc) < 1% readily measurable. The voltammetric signature of FcC(12)SH label is also able to differentiate types of defect in a given SAM. A number of SAM preparation conditions are assessed for the density and type of coverage defect. This labeling method therefore will be a useful tool for research into SAM property-function relationships.     

Influence of perfluorinated compounds on the properties of model lipid membranes

The influence of selected perfluorinated compounds (PFCs), perfluorooctanoic acid (PFOA) or perfluorooctanesulfonic acid (PFOS), on the structure and organization of lipid membranes was investigated using model membranes-lipid monolayers and bilayers. The simplest model--a lipid monolayer--was studied at the air-water interface using the Langmuir-Blodgett technique with surface pressure and surface potential measurements. Lipid bilayers were characterized by NMR techniques and molecular dynamics simulations. Two phospholipids, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), characterized by different surface properties have been chosen as components of the model membranes. For a DPPC monolayer, a phase transition from the liquid-expanded state to the liquid-condensed state can be observed upon compression at room temperature, while a DMPC monolayer under the same conditions remains in the liquid-expanded state. For each of the two lipids, the presence of both PFOA and PFOS leads to the formation of a more fluidic layer at the air-water interface. Pulsed field gradient NMR measurements of the lateral diffusion coefficient (DL) of DMPC and PFOA in oriented bilayers reveal that, upon addition of PFOA to DMPC bilayers, DL of DMPC decreases for small amounts of PFOA, while larger additions produce an increased DL. The DL values of PFOA were found to be slightly larger than those for DMPC, probably as a consequence of the water solubility of PFOA. Furthermore, 31P and 2H NMR showed that the gel-liquid crystalline phase transition temperature decreased by the addition of PFOA for concentrations of 5 mol % and above, indicating a destabilizing effect of PFOA on the membranes. Deuterium order parameters of deuterated DMPC were found to increase slightly upon increasing the PFOA concentration. The monolayer experiments reveal that PFOS also penetrates slowly into already preformed lipid layers, leading to a change of their properties with time. These experimental observations are in qualitative agreement with the computational results obtained from the molecular dynamics simulations showing a slow migration of PFCs from the surrounding water phase into DPPC and DMPC bilayers.     

Surface planar bilayers of phospholipids used in protein membrane reconstitution: an atomic force microscopy study

In this work, using atomic force microscopy (AFM), we have studied the influence of the temperature on the properties of the surface planar bilayers (SPBs) formed with: (i) the total lipid extract of Escherichia coli; (ii) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPC) (1:1, mol/mol); and, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol-amine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). According to the height profile analysis we performed, the height of the SPBs of DMPC:POPC were temperature dependent. Separated domains were observed in the SPBs of the POPE:POPG mixture and the E. coli lipid extract. The implication of those domains for the correct insertion of membrane proteins into proteoliposomes is discussed.     

Dynamic properties of bicellar lipid mixtures observed by rheometry and quadrupole echo decay

In bicellar dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), the transition from isotropic reorientation to partial orientational order, on warming, is known to coincide with a sharp increase in viscosity. In this work, cone-and-plate rheometry, (2)H NMR spectroscopy, and quadrupole echo decay observations have been used to obtain new insights into the dynamics of phases observed in bicellar DMPC/DHPC mixtures. Samples with 25% of the DMPC component deuterated were used to correlate rheological measurements with phase behavior observed by (2)H NMR spectroscopy. Mixtures containing only normal DMPC (DMPC/DHPC) or only chain perdeuterated DMPC (DMPC-d(54)/DHPC) were used to refine rheology and quadrupole echo decay measurements respectively. The viscosity peaked at 4-9 Pa·s, just above the isotropic-to-nematic transition, and then dropped as samples were warmed through the nematic-to-lamellar transition. Quadrupole echo decay times above the nematic-to-lamellar transition were significantly longer than typically observed in the liquid crystalline phase of saturated lipid multilamellar vesicles. This may indicate a damping of slow bilayer undulations resulting from the coupling of opposite bilayer surfaces by DHPC-lined pores.     

Redox mediation at 11-mercaptoundecanoic acid self-assembled monolayers on gold

Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and digital simulation techniques were used to investigate quantitatively the mechanism of electron transfer (ET) through densely packed and well-ordered self-assembled monolayers (SAMs) of 11-mercaptoundecanoic acid on gold, either pristine or modified by physically adsorbed glucose oxidase (GOx). In the presence of ferrocenylmethanol (FcMeOH) as a redox mediator, ET kinetics involving either solution-phase hydrophilic redox probes such as [Fe(CN)6]3-/4- or surface-immobilized GOx is greatly accelerated: [Fe(CN)6]3-/4- undergoes diffusion-controlled ET, while the enzymatic electrochemical conversion of glucose to gluconolactone is efficiently sustained by FcMeOH. Analysis of the results, also including the digital simulation of CV and EIS data, showed the prevalence of an ET mechanism according to the so-called membrane model that comprises the permeation of the redox mediator within the SAM and the intermolecular ET to the redox probe located outside the monolayer. The analysis of the catalytic current generated at the GOx/SAM electrode in the presence of glucose and FcMeOH allowed the high surface protein coverage suggested by X-ray photoelectron spectroscopy (XPS) measurements to be confirmed.     

Surface thermodynamic properties of monolayers versus reconstitution of a membrane protein in solid-supported bilayers

Atomic force microscopy (AFM) was used to study the influence of a membrane protein, lactose permease of Escherichia coli (LacY), on the surface spreading behavior and the features of self-assembled phospholipids bilayers on mica. The miscibility of phospholipids used, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), was investigated by surface pressure area isotherm measurements at the air-water interface. A composition with an equimolar proportion of POPC and DMPC was used to form the liposomes. Surface layers formed with DMPC:POPC (0.5:0.5, mol/mol) or LacY reconstituted in proteoliposomes with the same phospholipid composition were imaged by using AFM. When lactose permease was reconstituted in DMPC:POPC (0.5:0.5, mol/mol), self-assembled structures that remained firmly adsorbed onto the mica surface were observed. These sheets had an irregular shape and their upper layer was more corrugated than that obtained for the phospholipid matrix.     

Characterization and control of lipid layer fluidity in hybrid bilayer membranes

The main gel-to-liquid-crystal (LC) phase transition temperature, T(m), of the distal lipid layer in hybrid bilayer membranes (HBMs) under water was investigated using vibrational sum frequency spectroscopy (VSFS). VSFS has unique sensitivity to order/disorder transitions in the lipid acyl chains and can determine T(m) for the lipid monolayers in HBMs. We recently reported the observation that T(m) is raised and the transition width is broadened for the overlying phospholipid monolayer in HBM systems formed on densely packed crystalline self-assembled monolayers (SAMs) as compared to that of vesicles in solution. In this report, we establish that T(m) for the lipid layer of HBMs can be controlled by proper choice of the SAM underlayer. The SAM underlayer of the HBM was systematically altered by using an alkane thiol, a saturated thiolipid, a mixed SAM of a saturated lipid-pyridine disulfide, and finally a mixed SAM of an unsaturated lipid-pyridine disulfide. T(m) was measured for two different chain length saturated phosphatidylcholine lipid overlayers on these SAMs. The values obtained show that Tm of the lipid layer of HBMs is sensitive to the composition and/or packing density of molecules in the underlying SAM.     

Multivalent dendrimers at molecular printboards: influence of dendrimer structure on binding strength and stoichiometry and their electrochemically induced desorption

A fundamental understanding of multivalency can have a profound influence on bottom-up nanofabrication. For this purpose, three different types of ferrocenyl (Fc) functionalized dendrimers of generations 1-5 with various spacer groups were adsorbed at self-assembled monolayers (SAMs) of heptathioether-functionalized beta-cyclodextrin (betaCD) on gold. The dendrimers formed kinetically stable supramolecular assemblies at the betaCD host surface having up to eight multivalent supramolecular interactions, but could be efficiently removed from the host surface by electrochemical oxidation of the Fc end groups. Dendrimer desorption and re-adsorption could be repeated a number of times without significant decomposition of the system. The stoichiometries of the dendrimers at the surface were determined using cyclic voltammetry (CV). These were quantitatively confirmed for the lower generations by surface plasmon resonance (SPR) titrations of the dendrimers to the betaCD SAM. Measuring CV and SPR simultaneously gave crucial mechanistic information on the electrochemically induced desorption of the dendrimers from the host surface. The redox-active dendrimers effectively blocked the host surface for binding other molecules, e.g. adamantyl-functionalized dendrimers, but electrochemically induced release of the blocking layer revealed the host surface to which the adamantyl dendrimers could then bind.     

Interaction of the cationic peptide bactenecin with mixed phospholipid monolayers at the air-water interface

The initial mechanism by which antimicrobial peptides target microbes occurs via electrostatic interactions; however, the mechanism is not well understood. We investigate the interaction of the antimicrobial peptide bactenecin with a 50:50 w:w% 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG) phospholipid mixture at the air-water interface with different NaCl concentrations (0.01, 0.05, 0.1, 0.5 M) in the subphase. A larger shift of DPPC:DMPG isotherms was obtained for 0.1 M salt concentration at lower and higher pressures, demonstrating the influence of the negative charge of DMPG molecules and the screening of the electrostatic interaction by the salt concentration. Raman spectroscopy of monolayers demonstrated the presence of cysteine-cysteine bridges in bactenecin loops. The peptide adsorption in DPPC:DMPG monolayers observed by AFM images suggests a self-assembled aggregation process, starting with filament-like networks. Domains similar to carpets were formed and pore structures were obtained after a critical peptide concentration, according to the carpet model.     

Temperature driven annealing of perforations in bicellar model membranes

Bicellar model membranes composed of 1,2-dimyristoylphosphatidylcholine (DMPC) and 1,2-dihexanoylphosphatidylcholine (DHPC), with a DMPC/DHPC molar ratio of 5, and doped with the negatively charged lipid 1,2-dimyristoylphosphatidylglycerol (DMPG), at DMPG/DMPC molar ratios of 0.02 or 0.1, were examined using small angle neutron scattering (SANS), (31)P NMR, and (1)H pulsed field gradient (PFG) diffusion NMR with the goal of understanding temperature effects on the DHPC-dependent perforations in these self-assembled membrane mimetics. Over the temperature range studied via SANS (300-330 K), these bicellar lipid mixtures exhibited a well-ordered lamellar phase. The interlamellar spacing d increased with increasing temperature, in direct contrast to the decrease in d observed upon increasing temperature with otherwise identical lipid mixtures lacking DHPC. (31)P NMR measurements on magnetically aligned bicellar mixtures of identical composition indicated a progressive migration of DHPC from regions of high curvature into planar regions with increasing temperature, and in accord with the "mixed bicelle model" (Triba, M. N.; Warschawski, D. E.; Devaux, P. E. Biophys. J.2005, 88, 1887-1901). Parallel PFG diffusion NMR measurements of transbilayer water diffusion, where the observed diffusion is dependent on the fractional surface area of lamellar perforations, showed that transbilayer water diffusion decreased with increasing temperature. A model is proposed consistent with the SANS, (31)P NMR, and PFG diffusion NMR data, wherein increasing temperature drives the progressive migration of DHPC out of high-curvature regions, consequently decreasing the fractional volume of lamellar perforations, so that water occupying these perforations redistributes into the interlamellar volume, thereby increasing the interlamellar spacing.     

Time-dependent organization and wettability of decanethiol self-assembled monolayer on Au(111) investigated with STM

A detailed study on the time-dependent organization of a decanethiol self-assembled monolayer (SAM) at a designed solution concentration onto a Au(111) surface has been performed with scanning tunneling microscopy (STM). The SAMs were prepared by immersing Au(111) into an ethanol solution containing 1 microM decanethiol with different immersion times. STM images revealed the formation process and adlayer structure of the SAMs. It was found that the molecules self-organized into adlayers from random separation to a well-defined structure. From 10 s, small domains with ordered molecular organization appeared, although random molecules could be observed on Au(111) at the very initial stage. At 30 s, the SAM consisted of uniform short stripes. Each stripe consisted of sets of decanethiol mainly containing eight molecules. With the immersion time increasing, the length of the stripes increased. At 5 min, the alkyl chains overlapped each other between the adjacent stripes, indicating the start of a stacked process. After immersing Au(111) in decanethiol solution for 3 days, a densely packed adlayer with a (radical 3 x radical 3)R30 degrees structure was observed. The formation process and structure of decanethiol SAMs are well related to sample preparation conditions. The wettability of the decanethiolate SAM-modified Au(111) surface was also investigated.     

Interfacial properties of a synthetic peptide derived from hepatitis G virus E2 protein: interaction with lipid monolayers

A useful approach to get information about the potential fusogenic ability of virus synthetic peptides is the study of its interfacial properties and subsequent study in mono- and bilayers. In this work, we have characterized by means of physicochemical tools (i.e. compression isotherms and surface activity) the sequence 267-284, LLGTEVSEVLGGAGLTGG, derived from the E2 structural protein of HGV/GBV-C. The adsorption of the peptide at the air/water interface was monitored by following the increase in surface pressure as a function of time at two different pH values: 5 and 7. Parameters such as surface excess or molecular area were calculated from the equation of Gibbs. The peptide showed a tendency to migrate to the surface of a saline-buffered solution. It formed stable monolayers at the air/water interface giving a compression isotherm with a shape consistent with that of some alpha-helical peptide conformations. Brewster angle microscopy (BAM) showed that through compression the peptide formed multilayers. The studies with lipid monolayers (DPMC, DMPC/DMPG, and DMPC/DMTAP) showed that the peptide interacts with all the lipids assayed producing a marked disrupting effect upon them. In these effects electrostatic interactions seem to have some participation.