Effect of molecular surface packing on the enzymatic activity modulation of an anchored protein on phospholipid Langmuir monolayers

The catalytic activity of a glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase has been studied in Langmuir phospholipid monolayers at different surface pressures. The enzyme substrate, p-nitrophenyl phosphate, was injected into the subphase of mixed enzyme/lipid Langmuir monolayers. Its hydrolysis product was followed by monitoring the absorbance at 410 nm in situ in the monolayer subphase of the Langmuir trough. Several surface pressures, corresponding to different molecular surface densities, were attained by lateral compression of the monolayers. The morphology of the monolayers, observed by fluorescence microscopy, showed three different types of domains owing to the heterogeneous partition of the enzyme within the mixed enzyme/lipid film. The catalytic activity was modulated by the enzyme surface density, and it increased until a pressure of 18 mN/m was reached, but it decreased significantly when the equilibrium in-plane elasticity (surface compressional modulus) increased more noticeably, resulting in alterations in the interface morphology. A model for the modulation of the enzyme orientation and catalytic activity by lipid/enzyme surface morphology and enzyme surface packing at the air/liquid interface is proposed. The results might have an important impact on the comprehension of the enzymatic activity regulation of GPI-anchored proteins in biomembranes.     

Properties of oxidized phospholipid monolayers: An atomistic molecular dynamics study

Non-saturated lipids in the lung surfactant are prone to oxidation by oxidative species present in air. In this Letter, oxidized monolayers of dioleoylphosphatidylcholine (DOPC) were studied by means of both molecular dynamics and potential of mean force calculations. Structural changes of the monolayer following oxidation were revealed, including orientational reversal of oxidized chains. Accumulation of oxidized lipids in the monolayer is observed for moderate oxidation ratios, whereas removal of the short-chain oxidation product is predicted at long timescales. Massive oxidation leads to the loss of the well-ordered monolayer structure and partial solubilization of the oxidized lipids in the aqueous subphase.     

Grazing incidence X-ray diffraction studies of condensed double-chain phospholipid monolayers formed at the soft air/water interface

The use of highly brilliant synchrotron light sources in the middle of the 1980s for X-ray diffraction has revolutionized the research of condensed monolayers. Since then, monolayers gained popularity as convenient quasi two-dimensional model systems widely used in biophysics and material science. This review focuses on structures observed in one-component phospholipid monolayers used as simplified two-dimensional models of biological membranes. In a monolayer system the phase transitions can be easily triggered at constant temperature by increasing the packing density of the lipids by compression. Simultaneously the monolayer structure changes are followed in situ by grazing incidence X-ray diffraction. Competing interactions between the different parts of the molecule are responsible for the different monolayer structures. These forces can be modified by chemical variations of the hydrophobic chain region, of the hydrophilic head group region or of the interfacial region between chains and head groups. Modifications of monolayer structures triggered by changes of the chemical structure of double-chain phospholipids are highlighted in this paper.     

Nanometre-sized molecular oxygen sensors prepared from polymer stabilized phospholipid vesicles

Nanometre-sized, chemically-stabilized phospholipid vesicle sensors have been developed for detection of dissolved molecular oxygen. Sensors were prepared by forming 150 nm phospholipid vesicles from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or DOPC doped with small (<1%) mole percentages of 1,2-dioleoyl-sn-glycero-3-phosphoethanol amine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE). Sensors were stabilized via cross-linking polymerization of hydrophobic methacrylate monomers partitioned into the hydrophobic interior of the DOPC bilayer. The resultant unilamellar, nanometre-sized, polymer-lipid vesicles are spherical, biocompatible and protect sensing components that are loaded into the aqueous interior of the vesicle from interfering species in the exterior environment. For O(2) detection, the oxygen-sensitive fluorescent dye, tris(1,10-phenanthroline)ruthenium(II) chloride (Ru(phen)(3)) was encapsulated into the aqueous interior of the polymerized phospholipid vesicle. NBD-PE was introduced into the phospholipid bilayer of the sensor as a reference dye, allowing ratiometric sensors to be constructed. The resultant sensors show high sensitivity, excellent reversibility and excellent linearity over a physiological range of dissolved oxygen concentrations. These results suggest that polymerized phospholipid vesicle sensors can be used for monitoring intracellular O(2) dynamics.     

An LC method for monitoring medium-chain fatty acid permeation through CaCo-2 cell monolayers

A simple method was developed for monitoring the permeation of medium-chain fatty acids of C8 (octanoic acid) and C10 (decanoic acid) through CaCo-2 cell monolayers by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). The detection was made based on the electrochemical reduction prepeak of quinone caused by acids, requiring the fabrication of a two-channel HPLC-ECD system. In one channel, acetonitrile–water (7:3, v/v) was used as a mobile phase to separate acids by a C30 column. In the other channel, acetonitrile–water (7:3, v/v) containing 6 mmol/L 3,5-di-t-butyl-1,2-benzoquinone and 20 mmol/L LiClO₄ was used as a quinone solution to detect acids by an electrochemical cell with a glassy carbon working electrode. In this HPLC-ECD system, eluted acids were mixed with the quinone solution in a post column fashion to obtain current signals caused by acids. The peak area was found to be linearly related to the acid amount ranging from 25 to 1,000 pmol (r > 0.992). The detection limits of octanoic acid and decanoic acid were 7.5 and 8.8 pmol, respectively. Octanoic acid and decanoic acid spiked into cell culture media samples were extracted with acetonitrile and their recoveries were more than 89.5% with an RSD of less than 8.2%. This method was applied to the permeation experiment of octanoic acid and decanoic acid with CaCo-2 cell monolayers formed on the Transwell? system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was partly presented at The Physical Pharma Forum 2008 on March 24, 2008 in Tokyo, Japan.     

Effect of hydrocarbon chain and pH on structural and topographical characteristics of phospholipid monolayers

Structural characteristics (structure, elasticity, topography, and film thickness) of dipalmitoyl phosphatidylcholine (DPPC) and dioleoyl phosphatidylcholine (DOPC) monolayers were determined at the air-water interface at 20 degrees C and pH values of 5, 7, and 9 by means of surface pressure (pi)-area (A) isotherms combined with Brewster angle microscopy (BAM) and atomic force microscopy (AFM). From the pi-A isotherms and the monolayer elasticity, we deduced that, during compression, DPPC monolayers present a structural polymorphism at the air-water interface, with the homogeneous liquid-expanded (LE) structure; the liquid-condensed structure (LC) showing film anisotropy and DPPC domains with heterogeneous structures; and, finally, a homogeneous structure when the close-packed film molecules were in the solid (S) structure at higher surface pressures. However, DOPC monolayers had a liquid-expanded (LE) structure under all experimental conditions, a consequence of weak molecular interactions because of the double bond of the hydrocarbon chain. DPPC and DOPC monolayer structures are practically the same at pH values of 5 and 7, but a more expanded structure in the monolayer with a lower elasticity was observed at pH 9. BAM and AFM images corroborate, at the microscopic and nanoscopic levels, respectively, the same structural polymorphism deduced from the pi-A isotherm for DPPC and the homogeneous structure for DOPC monolayers as a function of surface pressure and the aqueous-phase pH. The results also corroborate that the structural characteristics and topography of phospholipids (DPPC and DOPC) are highly dependent on the presence of a double bond in the hydrocarbon chain.     

Headgroup percolation and collapse of condensed langmuir monolayers

We present a study of Langmuir isotherms and 2D bulk moduli of binary lipid mixtures, where changes in monolayer collapse pressure (Pic) are followed while varying the relative amounts of the two components. For monolayers containing dipalmitoylphosphocholine (DPPC) with either hexadecanol (HD) or palmitic acid (PA), a distinctly non-monotonic change in Pic is observed with varying composition. At low mole fractions, there is a slight decrease in Pic as films get richer in DPPC, while a sharp increase to pure DPPC-like values is observed when the mole fraction exceeds approximately 0.7. The sudden transition in collapse pressure is explained using the principles of rigidity percolation, and important ramifications of this phenomenon for biological surfactant are discussed.     

Modulated pattern formation of phospholipid monolayers on curved surfaces

Langmuir-Blodgett transfer of a dipalmitoylphosphatidylcholine monolayer onto macroscopically curved mica surfaces results in microscopic patterns of the transferred monolayer that differ from those of films transferred onto a flat mica substrate. On curved surfaces a modulated horizontal striped pattern evolves that has a zigzag boundary at the liquid condensed front of the stripe and a continuous straight boundary at the liquid condensed rear. We propose that the sensitivity of the pattern to the macroscopic curvature of the sample is due to a flow-controlled hydrodynamic instability caused by the subphase flow close to the three-phase contact line.     

Interaction of gramicidin derivatives with phospholipid monolayers

A study of the interaction of gramicidin A (gA), tert-butyloxycarbonyl-gramicidin (g-BOC), and desformyl gramicidin (g-des) with dioleoyl phosphatidylcholine (DOPC) and DOPC/phosphatidylserine (PS) mixed monolayers on a mercury electrode is reported in this paper. Experiments were carried out in electrolytes KCl (0.1 mol dm(-3)) and Mg(NO3)2 (0.05 mol dm(-3)). The channel-forming properties of the gramicidins were studied by following the reduction of Tl(I) to Tl(Hg). The frequency dependence of the complex impedance of coated electrode surfaces in the presence and absence of the gramicidins was estimated between 65,000 and 0.1 Hz at potentials of -0.4 V versus Ag/AgCl with 3.5 mol dm(-3) KCl. Epifluorescence microscopy was used to qualitatively correlate the interaction of the gramicidin peptides with dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylglycerol (DPPG) at the air-water interface. gA was shown to form Tl+ conducting channels in a DOPC monolayer, while g-BOC and g-des did not. In DOPC-30% PS (DOPC-0.3PS) layers, there is a marked increase in channel activity of all three gramicidin derivatives. None of the peptides facilitate the permeability of the DOPC-0.3PS layer to Cd2+. All three peptides interact with the layer as shown by capacitance-potential curves and impedance spectroscopy indicated by penetration of the peptide into the dielectric, an increase in surface "roughness", and an increased significance of low-frequency relaxations. The order of interaction is gA > g-des > g-BOC. The epifluorescence study of DPPC and DPPG layers at the air-water interface shows a selective action of the different gramicidins.     

Compressibility study of quaternary phospholipid blend monolayers

The mechanical properties of liposome membranes are strongly dependent on type and ratio of lipid compounds, which can have important role in drug targeting and release processes when liposome is used as drug carrier. In this work we have used Brewster's angle microscopy to monitor the lateral compression process of lipid monolayers containing as helper lipids either distearoyl phosphatidylethanolamine (DSPE) or dioleoyl phophatidylethanolamine (DOPE) molecules on the Langmuir trough. The compressibility coefficient was determined for lipid blend monolayers containing the helper lipids above, cholesterol, distearoyl phosphatidylcholine (DSPC) and pegylated-DSPE at room temperature. Two variables, the cholesterol fraction and the ratio ρ between the helper lipid (either DSPE or DOPE) and the reference lipid DSPC, were studied by multivariate analysis to evaluate their impact on the compressibility coefficient of the monolayers. The cholesterol level was found to be the most significant variable for DSPE blends while the ratio ρ was the most significant one for DOPE blend monolayers. It was also found that these two variables can exhibit positive interaction and the same compressibility value can be obtained with different blend compositions.     

Generic phase behavior of branched-chain phospholipid monolayers

Monolayers of chemically modified triple-chain phospholipids have been investigated at the air/water interface using pressure-area isotherms. The condensed phases of the lipids were characterized by grazing incidence X-ray diffraction (GIXD). Increasing chain length corresponds to a temperature effect, which was quantified for different lipids, depending on the head group structure using isotherm (two-dimensional systems) and DSC (three-dimensional systems) measurements. The combination of structure investigations revealed generic phase diagrams, which describe the phase behavior of multiple-chain lipids in two dimensions. For the 1-acyl-2-O-alkyl phospholipids, the generic phase diagram exhibits only L(2d), LS and LE phases while the exchange of the position of the branched acyl and the non-branched alkyl chains at the glycerol backbone leads to a much richer polymorphism (L(2h), L(2d), Ov, LS, S, tau, LE). Here we present the first experimental evidence of the unusual tau phase for multiple-chain lipid monolayers. This phase exhibits an undistorted in-plane lattice despite of tilted chains.     

Effect of membrane environment on proton permeation through gramicidin A channels

Multistate empirical valence bond simulations were employed to study proton transport through gramicidin A channels embedded in two different lipid bilayers, glycerol 1-monooleate (GMO) and diphytanolphosphatidylcholine (DiPhPC). Free energy barriers to proton permeation were derived using a new internal reaction coordinate describing the proton permeation process. The large quantitative and qualitative differences between the two systems are discussed in terms of local bilayer structures, ordering of interfacial water, and channel flexibility in the two environments.     

Electroreduction of oxygen on octadecylmercaptan self-assembled monolayers

The reduction of oxygen has been studied on octadecylmercaptan self-assembled monolayers adsorbed on gold substrates in borate buffer solutions with a rotating disc electrode. A great inhibition of the oxygen reduction and other electrochemical reactions by these monolayers has been found. However, after polarisation at –0.80 V_SHE the protecting properties of the film against electron transfer reactions are lost, and a behaviour similar to bare gold is observed. Ex situ XPS indicates that the thiol monolayer has not been desorbed to a large extent during oxygen reduction. Disorders of the monolayer structure and desorption of thiol molecules are proposed as the main reasons for the accessibility of electrochemical reactions to the surface.     

Electroreduction of oxygen on octadecylmercaptan self-assembled monolayers

The reduction of oxygen has been studied on octadecylmercaptan self-assembled monolayers adsorbed on gold substrates in borate buffer solutions with a rotating disc electrode. A great inhibition of the oxygen reduction and other electrochemical reactions by these monolayers has been found. However, after polarisation at -0.80 V(SHE) the protecting properties of the film against electron transfer reactions are lost, and a behaviour similar to bare gold is observed. Ex situ XPS indicates that the thiol monolayer has not been desorbed to a large extent during oxygen reduction. Disorders of the monolayer structure and desorption of thiol molecules are proposed as the main reasons for the accessibility of electrochemical reactions to the surface.     

Charge transport through self-assembled monolayers of compounds of interest in molecular electronics

The electrical properties of self-assembled monolayers (SAMs) on metal surfaces have been explored for a series of molecules to address the relation between the behavior of a molecule and its structure. We probed interfacial electron transfer processes, particularly those involving unoccupied states, of SAMs of thiolates or arylates on Au by using shear force-based scanning probe microscopy (SPM) combined with current-voltage (i-V) and current-distance (i-d) measurements. The i-V curves of hexadecanethiol in the low bias regime were symmetric around 0 V and the current increased exponentially with V at high bias voltage. Different than hexadecanethiol, reversible peak-shaped i-V characteristics were obtained for most of the nitro-based oligo(phenylene ethynylene) SAMs studied here, indicating that part of the conduction mechanism of these junctions involved resonance tunneling. These reversible peaked i-V curves, often described as a negative differential resistance (NDR) effect of the junction, can be used to define a threshold tip bias, V(TH), for resonant conduction. We also found that for all of the SAMs studied here, the current decreased with increasing distance, d, between tip and substrate. The attenuation factor beta of hexadecanethiol was high, ranging from 1.3 to 1.4 A(-1), and was nearly independent of the tip bias. The beta-values for nitro-based molecules were low and depended strongly on the tip bias, ranging from 0.15 A(-1) for tetranitro oligo(phenylene ethynylene) thiol, VII, to 0.50 A(-1) for dinitro oligo(phenylene) thiol, VI, at a -3.0 V tip bias. Both the V(TH) and beta values of these nitro-based SAMs were also strongly dependent on the structures of the molecules, e.g. the number of electroactive substituent groups on the central benzene, the molecular wire backbone, the anchoring linkage, and the headgroup. We also observed charge storage on nitro-based molecules. For a SAM of the dintro compound, V, approximately 25% of charge collected in the negative scan is stored in the molecules and can be collected at positive voltages. A possible mechanism involving lateral electron hopping is proposed to explain this phenomenon.     

Coarse-grained nonequilibrium approach to the molecular modeling of permeation through microporous membranes

We present a modeling technique that combines a statistical-mechanical coarse-graining scheme with a nonequilibrium molecular simulation algorithm to provide an efficient simulation of steady-state permeation across a microporous material. The coarse-graining scheme is based on the mapping of an atomistic model to a lattice using multidimensional free-energy and transition-state calculations. The nonequilibrium simulation algorithm is a stochastic, lattice version of the recently developed atomistic dual-control-volume grand canonical molecular dynamics. We demonstrate the approach on a model of methane permeating through a bulk portion of siliceous zeolite ZK4 at 300 K under imposed fugacity differences. We predict the coarse-grained (cage-level) density profiles and observe the development of nonlinearities as the magnitude of the fugacity difference is increased. From the net flux of methane we also predict a mean permeability coefficient under the various conditions. The simulation results are obtained over time scales on the order of microseconds and length scales on the order of dozens of nanometers.     

Electrons are transported through phenylene-ethynylene oligomer monolayers via localized molecular orbitals

We report excellent correlations between the first negative threshold potentials (V(TH)s) for electric conduction, electrochemical potentials, and computed lowest unoccupied molecular orbital energies in a series of phenylene-ethynylene oligomers bearing a sulfur-based anchoring unit and different electroactive substituents on the central benzene ring. The theoretical and electrochemical results strongly suggest that the peaks observed in the i-V curves have a true molecular origin and are associated with distinct unoccupied molecular levels of the compounds that are strongly localized on the central ring (except for compound I). This localization might account for the existence of a long-lived radical-anion state that permits lateral electron hopping and leads to charge trapping and storage.