Electrochemical investigations of cholesterol enriched glassy carbon supported thin lipid films

The formation and study of stable cholesterol enriched thin lipid layers onto the surface of glassy carbon electrode is reported in this work. The method of formation relies on additional thinning of wetting films by electrostriction. Electrochemical techniques based on the concepts of impedance and voltammetry are used to explore the films' features. The impedance data reveal a substantial change of relaxation characteristics of the modified films. In this respect, opportunities for the evaluation of the films' stage based on the approximation with 'constant phase angle element' are discussed. The possible final structure of the films, as well as, their relevance for development of sensor elements are briefly viewed.     

Lipid mono- and bilayer supported on polymer films: composite polymer-lipid films on solid substrates.

We report the deposition of lipid monolayers and bilayers on polyacrylamide films deposited by radical chain reaction onto solid substrates in aqueous solutions. Polymer films of various degrees of monomer density and cross-linking are prepared. Lateral diffusion and fluorescent probe permeation measurements yield insight into the continuity of the lipid layers and show that monolayers exposed to air are much less sensitive towards polymer heterogeneities than bilayers below water, which is explained in terms of the wetting laws. The diffusion studies of lipid and lipopeptide probes yield absolute values of the frictional coefficients between the lipid layer and the polymer films and allow one to estimate the surface viscosity of the polymer film. The potential applications of supported membranes on soft thin polymer films for the preparation of biofunctionalized surfaces or biocompatible receptive surfaces for biosensors are discussed.     

Structural dynamics of a lytic peptide interacting with a supported lipid bilayer

The interaction of a melittin mutant with a 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)-supported lipid bilayer was studied with the use of time-resolved evanescent wave-induced fluorescence spectroscopy (TREWIFS) and evanescent wave-induced time-resolved fluorescence anisotropy measurements (EW-TRAMs). The mutant peptide was labeled at position K14 with AlexaFluor 430 and retained the lytic activity characteristic of native melittin. The fluorescence decay kinetics of the conjugate was found to be biexponential with a short-lived component, τ₁, due to photoinduced electron transfer between AlexaFluor 430 and proximal side chains within or between the peptides. The longer-lived component, τ₂, was sensitive to the polarity of the microenvironment at or near the K14 position of the peptide. Upon interaction with a DPPC-supported bilayer, the proportional contribution of τ₁ increased, indicating a conformational change of the peptide. The values of τ₁ and τ₂ indicate that the AlexaFluor 430 probe experienced an environment with an equivalent polarity no less than that of methanol. EW-TRAMs data from the melittin mutant revealed hindered rotational motions of the AlexaFluor 430 probe both in the plane and perpendicular to the plane of the supported lipid bilayer. The data indicate a highly ordered and polar environment near the center of the melittin helix consistent with the formation of a toroidal pore.     

Advances in the characterization of supported lipid films with the atomic force microscope

During the past decade, the atomic force microscope (AFM) has become a key technique in biochemistry and biophysics to characterize supported lipid films, as testified by the continuous growth in the number of papers published in the field. The unique capabilities of AFM are: (i) capacity to probe, in real time and in aqueous environment, the surface structure of lipid films; (ii) ability to directly measure physical properties at high spatial resolution; (iii) possibility to modify the film structure and biophysical processes in a controlled way. Such experiments, published up to June 2000, are the focus of the present review. First, we provide a general introduction on the preparation and characterization of supported lipid films as well as on the principles of AFM. The section 'Structural properties' focuses on the various applications of AFM for characterizing the structure of supported lipid films: visualization of molecular structure, formation of structural defects, effect of external agents, formation of supported films, organization of phase-separated films (coexistence region, mixed films) and, finally, the use of supported lipid bilayers for anchoring biomolecules such as DNA, enzymes and crystalline protein arrays. The section 'Physical properties' introduces the principles of force measurements by AFM, interpretation of these measurements and their recent application to supported lipid films and related structures. Finally, we highlight the major achievements brought by the technique and some of the current limitations.     

Interaction of epithelial cells with the edges of fluid and solid lipid films

Capping of Concanavalin A (Con A) on the surface of epithelial cells near the cell-cell contacts has been compared with that in the regions of cell contacts with the edges of lipid films. If the lipids are in "fluid" state, Con A is capped likely as on the free edges of epithelial sheets, while contacts with the edge of solid lipid film inhibit capping of Con A as do cell-cell contacts. The same is true for capping of liposomes adsorbed on the surface of epithelial cells. We suppose that solid rather than fluid domains in plasma membranes may play a significant role in establishing cell-cell contacts.     

Recurrent dynamics of rupture transitions of giant lipid vesicles at solid surfaces

Single giant unilamellar vesicles (GUVs) rupture spontaneously from their salt-laden suspension onto solid surfaces. At hydrophobic surfaces, the GUVs rupture via a recurrent, bouncing ball rhythm. During each contact, the GUVs, rendered tense by the substrate interactions, porate, and spread a molecularly transformed motif of a monomolecular layer on the hydrophobic surface from the point of contact in a symmetric manner. The competition from pore closure, however, limits the spreading and produces a daughter vesicle, which re-engages with the substrate. At solid hydrophilic surfaces, by contrast, GUVs rupture via a distinctly different recurrent burst-heal dynamics; during burst, single pores nucleate at the contact boundary of the adhering vesicles, facilitating asymmetric spreading and producing a “heart”-shaped membrane patch. During the healing phase, the competing pore closure produces a daughter vesicle. In both cases, the pattern of burst-reseal events repeats multiple times, splashing and spreading the vesicular fragments as bilayer patches at the solid surface in a pulsatory manner. These remarkable recurrent dynamics arise, not because of the elastic properties of the solid surface, but because the competition between membrane spreading and pore healing, prompted by the surface-energy-dependent adhesion, determine the course of the topological transition.     

Painted supported lipid membranes

We report herein measurements on a novel type of supported lipid films, which we call painted supported membranes (PSM). These membranes are formed in a self-assembly process on alkylated gold films from an organic solution. The formation process was investigated with surface plasmon resonance microscopy. The optical and electrical properties of the films were determined for various types of lipids and as a function of temperature by means of cyclic voltammetry and potential relaxation after charge injection. We could show that these films exhibit an extraordinarily high specific resistivity which, depending on the lipid, may be as high as 10⁹ ohm/cm². We could also show that due to this low conductivity, an electrical polarization across the PSM relaxes with characteristic time constants of up to 20 min. The electrical properties together with their high mechanical stability and accessibility to surface sensitive techniques make these films well suitable model membranes for optical and electrical investigations. Examples for such applications are given in the subsequent article by Seifert et al.     

Electrostriction of vesicular membranes

Methods for consideration of membrane electrostriction existing in literature are critically analysed. Two cases of electrostriction are dealt with: the membrane is closed (vesicles), the membrane exchanges molecules with reservoir (BLM with meniscus). Deformations and elastic forces arising in uncharged vesicle with the constant number of particles when the electrostatic potential difference is applied across the membrane are calculated.     

Lipopeptides derived from HIV and SIV mimicking the prehairpin intermediate of gp41 on solid supported lipid bilayers

We present a universal mimetic approach of the prehairpin intermediate of gp41, which represents the active drug target for fusion inhibitors of HIV (human immunodeficiency virus) and SIV (simian immunodeficiency virus) based on membrane anchored lipopeptides. For this purpose, we have in situ coupled terminal cysteine-modified peptides originating from the NHR of SIV and HIV to a maleimide-functionalized DOPC bilayer and monitored the interactions with potential antagonists of the trimer-of-hairpin conformation C34 and T20 peptides by means of atomic force microscopy and ellipsometry. FT-IR analysis in conjugation with CD-spectroscopy of hydrated N36-lipopeptides, incorporated in multilamellar bilayer stacks was employed to investigate peptide conformation prior to antagonist binding. In contrast to solution studies substantial secondary structure formation of S-N36 after in situ coupling to the bilayer was found. We could show that S-N36-lipopeptide-aggregates in bilayers were selectively able to bind T20 or the corresponding C-peptides (C34) and similar results could be achieved by using H-N36 lipopeptides. It was found that T20 binding to coiled coil S-N36 lipopeptide assemblies was fully reversible at elevated temperatures, while T20 binds irreversibly to H-N36 bundles.     

Organization and dynamics of the proteolipid complexes formed by annexin V and lipids in planar supported lipid bilayers

The consequences of the binding of annexin V on its lateral mobility and that of lipids were investigated by means of experimental and simulated FRAP experiments. Experiments were carried out on planar supported bilayers (PC/PS 9:1 mol/mol mixtures) in the presence of 1 mM CaCl2 in the subphase. The probes C12-NBD-PS and fluorescein-labeled annexin V were used and the data compared with that previously obtained for C12-NBD-PC [Saurel, O., Cézanne, L., Milon, A., Tocanne, J. F., & Demange, P. (1998) Biochemistry 37, 1403-1410]. At complete coverage of the lipid bilayer by the protein (Cannexin = 80 nM), the lateral mobility of C12-NBD-PC was reduced by 40% while C12-NBD-PS and bound annexin V molecules were nearly immobilized (D < 10(-)11 cm2/s). At moderate protein concentration (20 nM < Cannexin < 80 nM), best fitting of the lipid and protein probe recoveries was achieved with one single diffusion coefficient and a mobile fraction close to 100%, indicating homogeneous lipid and protein populations. In contrast, at low protein concentration (Cannexin < 20 nM), C12-NBD-PS showed a two-component diffusion. The slow PS population at Cannexin < 20 nM and the single PS population at Cannexin > 20 nM moved at the same rate that bound annexin V (mobile fraction close to 100%), indicating strong PS/protein interactions. With the aid of computer simulations of the lateral motion of PC molecules, based on the 2-D crystalline networks formed by annexin V in contact with the lipid bilayer, these FRAP results may be accounted for by considering a rather simple model of a proteolipidic complex consisting of an extended 2-D crystalline protein network facing the lipid bilayer and stabilized by strong interactions between annexin V and PS molecules. In this model, immobilization of annexin V and PS molecules originates from their mutual interactions. The slowing down of PC molecules is due to various obstacles to their lateral diffusion which can be described as: the four PS molecules bound to the protein, the tryptophan 187 which presumably interacts with the lipids at the level of their polar headgroups and probably the three other hydrophobic amino acid residues located on the AB calcium-binding loops of the protein.     

Structure and dynamics of supported intermembrane junctions

Supported intermembrane junctions, formed by rupture of giant unilamellar vesicles onto conventional supported lipid membranes, have recently emerged as model systems for the study of biochemical processes at membrane interfaces. Using intermembrane fluorescence resonance energy transfer and optical standing wave fluorescence interferometry, we characterize the nanometer-scale topography of supported intermembrane junctions and find two distinct association states. In one state, the two membranes adhere in close apposition, with intermembrane separations of a few nanometers. In the second state, large intermembrane spacings of approximately 50 nm are maintained by a balance between Helfrich (entropic) repulsion and occasional sites of tight adhesion that pin the two membranes together. Reversible transitions between these two states can be triggered with temperature changes. We further examine the physical properties of membranes in each state using a membrane mixture near its miscibility phase transition temperature. Thermodynamic characteristics of the phase transition and diffusive mobility of individual lipids are comparable. However, collective Brownian motion of phase-separated domains and compositional fluctuations are substantially modulated by intermembrane spacing. The scaling properties of diffusion coefficient with particle size are determined from detailed analysis of domain motion in the different junction types. The results provide experimental verification of a theoretical model for two-dimensional mobility in membranes, which includes frictional coupling across an interstitial water layer.     

Functional incorporation of integrins into solid supported membranes on ultrathin films of cellulose: impact on adhesion

Biomimetic models of cell surfaces were designed to study the physical basis of cell adhesion. Vesicles bearing reconstituted blood platelet integrin receptors alpha(IIb)beta(3) were spread on ultrathin films of cellulose, forming continuous supported membranes. One fraction of the integrin receptors, which were facing their extracellular domain toward the aqueous phase, were mobile, exhibiting a diffusion constant of 0.6 micro m(2) s(-1). The functionality of receptors on bare glass and on cellulose cushions was compared by measuring adhesion strength to giant vesicles. The vesicles contained lipid-coupled cyclic hexapeptides that are specifically recognized by integrin alpha(IIb)beta(3). To mimic the steric repulsion forces of the cell glycocalix, lipids with polyethylene glycol headgroups were incorporated into the vesicles. The free adhesion energy per unit area deltag(ad) was determined by micro-interferometric analysis of the vesicle's contour near the membrane surface in terms of the equilibrium of the elastic forces. By accounting for the reduction of the adhesion strength by the repellers and from measuring the density of receptors one could estimate the specific receptor ligand binding energy. We estimate the receptor-ligand binding energy to be 10 k(B)T under bioanalogue conditions.     

Cellular lipid dynamics

During the past years, the notion of microdomains at the surface of cellular membranes has been developed. These are constituted by lipid rafts which involve sphingoglycolipids and cholesterol. To these rafts are associated proteins which have a lipid anchor or are transmembrane proteins. These lipid rafts target specific proteins at the plasma membrane surface and can remain associated with them. They are present in surface receptors and endocytosis occurs upon binding of the specific ligands. Thus these rafts participate to major aspects of cellular dynamics. These rafts are complex structures, insoluble in non-ionic detergents. According to the detergent used, many types of rafts can be isolated. Any alteration of cholesterol, sphingoglycolipids, or abnormalities of the proteins themselves, can lead to abnormal targeting at the membrane surface. It is possible that specific sphingoglycolipids are necessary to target specific proteins at the membrane surface. This may explain the complexity of the sphingoglycolipid molecules, both in relation to their oligosaccharide and to their ceramide structures. There is both a cellular and a tissue specificity of these constituents. Complex sphingoglycolipids are involved in cellular differentiation, cellular polarization, and modified in relation to cancer. Virus and bacteria can be linked to the sphingoglycolipids of these microdomains and alter cellular signaling and function. Sphingoglycolipids are involved in autoimmune diseases as antibody targets and in neurolipidoses which are genetic diseases involving their catabolism. The dynamics of the lipid rafts, in relation to cholesterol, can be altered in Niemann-Pick's disease type C and in Alzheimer's disease. Thus these microdomains are involved in many aspects related to normal and pathological cellular dynamics.     

Two-dimensional microelectrophoresis in supported lipid bilayers

We report the application of supported bilayers for two-dimensional microelectrophoresis. This method allows the lateral separation and accumulation of charged amphiphilic molecular probes in bilayers by application of an electric field parallel to the bilayer surface. Diffusion coefficient and mobility of the fluorescent probes are determined by observation of the fluorescence recovery after photobleaching (pattern bleaching). The diffusion coefficients and the mobilities of oppositely charged fluorescent probes in one bilayer can be determined independently from a single measurement. By analysis of the motion of charged and uncharged probes in one membrane one can distinguish between the motion caused by the electric field acting on the charge of individual probes and that caused by frictional forces due to electroosmosis.     

Raman spectra of planar supported lipid bilayers

Raman scattering has been used to obtain high quality vibrational spectra of planar supported lipid bilayers (pslb's) at the silica/water interface without the use of resonance or surface enhancement. A total internal reflection geometry was used both to increase the bilayer signal and to suppress the water background. Polarization control permits the determination of four components of the Raman tensor, of which three are independent for a uniaxial film. Spectra are reported of the phospholipids DMPC, DPPC, and POPC, in the C-H stretching region and the fingerprint region. The temperature-dependent polarized spectra of POPC show only small changes over the range 14-41 °C. The corresponding spectra of DMPC and DPPC bilayers show large thermal changes consistent with a decreasing tilt angle from the surface normal and increasing chain ordering at lower temperatures. The thermal behavior of DMPC pslb's is similar to that of vesicles of the same lipid in bulk suspension. In contrast to calorimetry, which shows a sharp phase transition (L_α-L_β') with decreasing temperature, the changes in the Raman spectra occur over a temperature range of ca. 10 °C commencing at the calorimetric phase transition temperature.     

Raman spectra of planar supported lipid bilayers

Raman scattering has been used to obtain high quality vibrational spectra of planar supported lipid bilayers (pslb's) at the silica/water interface without the use of resonance or surface enhancement. A total internal reflection geometry was used both to increase the bilayer signal and to suppress the water background. Polarization control permits the determination of four components of the Raman tensor, of which three are independent for a uniaxial film. Spectra are reported of the phospholipids DMPC, DPPC, and POPC, in the C-H stretching region and the fingerprint region. The temperature-dependent polarized spectra of POPC show only small changes over the range 14-41 degrees C. The corresponding spectra of DMPC and DPPC bilayers show large thermal changes consistent with a decreasing tilt angle from the surface normal and increasing chain ordering at lower temperatures. The thermal behavior of DMPC pslb's is similar to that of vesicles of the same lipid in bulk suspension. In contrast to calorimetry, which shows a sharp phase transition (L alpha-L beta') with decreasing temperature, the changes in the Raman spectra occur over a temperature range of ca. 10 degrees C commencing at the calorimetric phase transition temperature.     

Atomic force microscopy of supported lipid bilayers

Supported lipid bilayers (SLBs) are widely used in biophysical research to investigate the properties of biological membranes and offer exciting prospects in nanobiotechnology. Atomic force microscopy (AFM) has become a well-established technique for imaging SLBs at nanometer resolution. A unique feature of AFM is its ability to monitor dynamic processes, such as the interaction of bilayers with proteins and drugs. Here, we present protocols for preparing dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers supported on mica using small unilamellar vesicles and for imaging their nanoscale interaction with the antibiotic azithromycin using AFM. The entire protocol can be completed in 10 h.     

Phosphatidylethanolamine enhances rhodopsin photoactivation and transducin binding in a solid supported lipid bilayer as determined using plasmon-waveguide resonance spectroscopy

Flash photolysis studies have shown that the membrane lipid environment strongly influences the ability of rhodopsin to form the key metarhodopsin II intermediate. Here we have used plasmon-waveguide resonance (PWR) spectroscopy, an optical method sensitive to both mass and conformation, to probe the effects of lipid composition on conformational changes of rhodopsin induced by light and due to binding and activation of transducin (G(t)). Octylglucoside-solubilized rhodopsin was incorporated by detergent dilution into solid-supported bilayers composed either of egg phosphatidylcholine or various mixtures of a nonlamellar-forming lipid (dioleoylphosphatidylethanolamine; DOPE) together with a lamellar-forming lipid (dioleoylphosphatidylcholine; DOPC). Light-induced proteolipid conformational changes as a function of pH correlated well with previous flash photolysis studies, indicating that the PWR spectral shifts monitored metarhodopsin II formation. The magnitude of these effects, and hence the extent of the conformational transition, was found to be proportional to the DOPE content. Our data are consistent with previous suggestions that lipids having a negative spontaneous curvature favor elongation of rhodopsin during the activation process. In addition, measurements of the G(t)/rhodopsin interaction in a DOPC/DOPE (25:75) bilayer at pH 5 demonstrated that light activation increased the affinity for G(t) from 64 nM to 0.7 nM, whereas G(t) affinity for dark-adapted rhodopsin was unchanged. By contrast, in DOPC bilayers the affinity of G(t) for light-activated rhodopsin was only 18 nM at pH 5. Moreover exchange of GDP for GTP gamma S was also monitored by PWR spectroscopy. Only the light-activated receptor was able to induce this exchange which was unaffected by DOPE incorporation. These findings demonstrate that nonbilayer-forming lipids can alter functionally linked conformational changes of G-protein-coupled receptors in membranes, as well as their interactions with downstream effector proteins.     

Molecular transport and organization in supported lipid membranes

The mechanism by which vesicles spontaneously form supported lipid bilayer membranes on glass surfaces is becoming better understood and this knowledge is the basis of a technology of patterning membrane arrays and controlling composition. Controlled interactions between supported membranes and cells, particularly from the immune system, provide direct insight into cell-cell surface interactions.     

Specific binding of peanut agglutinin to GM1-doped solid supported lipid bilayers investigated by shear wave resonator measurements

This study deals with the specific interaction between the lectin peanut agglutinin (PNA) from Arachis hypogaea and the ganglioside G_M1 which was incorporated in a solid supported lipid bilayer immobilized on a gold electrode placed on top of an AT-cut quartz crystal. Bilayer formation was reached by self-assembly processes. The first monolayer consists of octanethiol attached to the gold surface via chemisorption and the second monolayer was immobilized by vesicle fusion on the preformed hydrophobic surface. We managed to keep unspecific binding to a minimum by using a phospholipid matrix consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Lectin binding to ganglioside G_M1 containing membranes was determined by a decrease of the resonant frequency of the quartz crystal. The minimum amount of receptor within the membrane which is necessary to obtain a complete protein monolayer was found to be less than 2 mol%. The adsorption isotherm of PNA to G_M1 was recorded and analyzed to be of Langmuir type, exhibiting a binding constant of PNA to the ganglioside of 8.3 ⋅ 10⁵ M^–1. The good agreement of the calculated Langmuir adsorption isotherm with the obtained experimental data implies that protein multilayers are not formed and that interactions between the adsorbents can be neglected. Furthermore, the association constants of two different saccharides, β-Galp-(1 → 3)-GalNAc exhibiting a strong binding to PNA in solution, and β-D-galactose with a much lower affinity were estimated by determining the equilibrium concentration of PNA attached to the surface. Moreover we were able to remove the attached lectin monolayer by digestion of the protein with pronase causing an increase in the resonant frequency which almost reversed the frequency shift to lower frequencies during adsorption. An even more complex system was built up by the use of digoxigenin-labeled PNA which also binds to the solid supported membrane containing the receptor G_M1. The immobilized lectin was recognized by anti-digoxigenin-F_ab-fragments, which is measurable by a further decrease of the resonant frequency. For all binding processes we found larger frequency shifts for a complete protein monolayer than predicted by Sauerbrey's equation, clearly showing that in addition to mass loading viscoelastic changes occur at the lipid-protein interface. Received: 22 July 1996 / Accepted: 12 September 1996