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.     

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.     

Fusion of liposomes with planar lipid bilayers

The fusion of liposomes with planar lipid bilayers was monitored by two different methods. (a) Liposomes consisting of phospholipids and cholesterol were added to the aqueous phase bathing the cholesterol-deficient planar lipid bilayers in the presence of nystatin. The resulting increase in the planar lipid bilayer's electrical conductance was considered indicative of fusion. (b) Transplanar lipid bilayer injection of ³⁵SO₄^2? trapped inside the liposomes.It is shown by both methods that fusion is specifically dependent on the presence of negatively charged phospholipids both in the liposomes and the planar lipid bilayers and on Ca²⁺ in the aqueous phase of the fusion system.     

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.     

Colicin E1 in planar lipid bilayers

The channel formed by the C-terminal domain of colicin E1 in planar lipid bilayers has proven to be more complex than one might have guessed for such a simple system. The protein undergoes a pH-dependent rearrangement which transforms it from a water soluble form to a much different membrane bound form. There are at least two bound states which don't form a channel. The process by which the channel opens and closes is regulated by the pH and the transmembrane voltage. The voltage is probably sensed by at least 3 (and more likely 4 or more) lysine residues which must be driven through the field to open the channel. The process appears to be hindered by particular carboxyl groups when they are in the unprotonated state. The open channel has several substates and several superstates. Very large positive voltage catalyzes a transition of the open channel to an inactivated state, and may be able to drive the channel-forming region of the protein across the membrane. Little is known about the structure of any of these states, but the open channel is large enough to allow NAD to traverse the membrane and appears to be formed by one colicin molecule. This single polypeptide mimics many of the properties found in channels of mammalian cell membranes, but it may prove more relevant as a model for the transport of proteins across membranes. The comparative ease with which the protein can be manipulated chemically and genetically, along with the complexity of its behavior, promises to keep several laboratories busy for some time.     

Atomic force microscopy of supported planar membrane bilayers.

Membrane bilayers of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE) adsorbed to a freshly cleaved mica substrate have been imaged by Atomic Force Microscopy (AFM). The membranes were mounted for imaging by two methods: (a) by dialysis of a detergent solution of the lipid in the presence of the substrate material, and (b) by adsorption of lipid vesicles onto the substrate surface from a vesicle suspension. The images were taken in air, and show lipid bilayers adhering to the surface either in isolated patches or in continuous sheets, depending on the deposition conditions. Epifluorescence light-microscopy shows that the lipid is distributed on the substrate surfaces as seen in the AFM images. In some instances, when DPPE was used, whole, unfused vesicles, which were bound to the substrate, could be imaged by the AFM. Such membranes should be capable of acting as natural anchors for imaging membrane proteins by AFM.     

Fluorescence-quenching-resolved spectra of melittin in lipid bilayers

The interaction of bee venom melittin with dimyristoylphosphatidylcholine (DMPC) unilamellar vesicles has been studied by means of fluorescence quenching of the single tryptophan residue of the protein, at lipid-to-peptide ratio, Ri = 50 and at high ionic strength (2 M NaCl). The method of fluorescence-quenching-resolved spectra (FQRS), applied in this study with potassium iodide as a quencher, enabled us to decompose the tryptophan emission spectrum of liposome-bound melittin into components, at temperatures above as well as below the main phase transition temperature (Tt) of DMPC. One of the two resolved spectra exhibits maximum at 342 and 338 nm for experiments above and below Tt, respectively, and is similar to the maximum of tryptophan emission found for tetrameric melittin in solution (340 nm). This spectrum is characterized by the Stern-Volmer quenching constant, Ksv, of about 4 M-1 and it represents the fraction of melittin molecules whose tryptophan residues are exposed to the solvent to a degree comparable with tetrameric species in solution. The other spectrum component, corresponding to the quencher-inaccessible fraction of tryptophan molecules (Ksv = 0 M-1) has its maximum blue-shifted up to 15 nm, indicating a decrease in polarity of the environment. For experiments above Tt, the blue spectrum component revealed the excitation-wavelength dependence, originating probably from the relaxation processes between the excited tryptophan molecules and lipid polar head groups. We conclude that melittin bound to DMPC liposomes exists in two lipid-associated forms; one, with tryptophan residues exposed to the solvent and the other, penetrating the membrane interior, with tryptophan residues located in close proximity to the phospholipid polar head groups of the outer vesicle lipid layer. We also discuss our data with current models of melittin-bilayer interactions.     

Electric field-induced concentration gradients in planar supported bilayers.

A simple method of generating electric field-induced concentration gradients in planar supported bilayers has been developed. Gradients of charged, fluorescently labeled probes were visualized by epifluorescence microscopy and could be observed at field strengths as low as 1 V/cm. Steady-state concentration gradients can be described by a simple competition between random diffusion and electric field-induced drift. A model based on this principle has been used to determine the diffusion coefficient of the fluorescent probes. This technique achieves a degree of electrical manipulation of supported bilayers that offers a variety of possibilities for the development of new molecular architectures and the study of biological membranes.     

Formation of three-dimensional structures in supported lipid bilayers

The creation of three-dimensional structures in supported lipid bilayers has been examined. In bilayers, shape transformations can be triggered by adjusting a variety of parameters. Here, it is shown that bilayers composed of phosphatidylcholine and phosphatidic acid can be induced to reversibly form cap structures when exposed to an asymmetry in ionic strength. The structures that form depend on the asymmetry in the ionic strength and the amount of anionic lipid. Other factors that may be of importance in the creation of the structures, expansion forces, osmotic forces, and the bilayer-support interaction are discussed. The cap structures have the potential to be of considerable utility in examining the effect that curvature has on membrane processes.     

Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers.

A technique for the production of supported phospholipid bilayers by adsorption and fusion of small unilamellar vesicles to supported phospholipid monolayers on quartz is described. The physical properties of these supported bilayers are compared with those of supported bilayers which are prepared by Langmuir-Blodgett deposition or by direct vesicle fusion to plain quartz slides. The time courses of vesicle adsorption, fusion and desorption are followed by total internal reflection fluorescence microscopy and the lateral diffusion of the lipids in the adsorbed layers by fluorescence recovery after photobleaching. Complete supported bilayers can be formed with phosphatidylcholine vesicles at concentrations as low as 35 microM. However, the adsorption, fusion and desorption kinetics strongly depend on the used lipid, NaCl and Ca2+ concentrations. Asymmetric negatively charged supported bilayers can be produced by incubating a phosphatidylcholine monolayer with vesicles composed of 80% phosphatidylcholine and 20% phosphatidylglycerol. Adsorbed vesicles can be removed by washing with buffer. The measured fluorescence intensities after washing are consistent with single supported bilayers. The lateral diffusion experiments confirm that continuous extended bilayers are formed by the monolayer-fusion technique. The measured lateral diffusion coefficient of NBD-labeled phosphatidylethanolamine is (3.6 +/- 0.5) x 10(-8) cm2/s in supported phosphatidylcholine bilayers, independent of the method by which the bilayers were prepared.     

Interactions of Folch-Lees proteolipid apoprotein with planar lipid bilayers

Summary Water-soluble Folch-Lees proteolipid apoprotein from bovine CNS white matter induces a voltage-dependent conductance in black lipid membranes. Na⁺ is required for the induced conductance change but the established conductance has very low ionic selectivity. The induced conductance fluctuates with a minimum amplitude of 10^–11–10^–10 mho. The magnitude of the conductivity change is dependent on protein concentration and on the composition of lipid bilayers. At a fixed voltage the induced conductance of a phosphatidylcholine-cholesterol membrane is proportional to the sixth power of the protein concentration and the first power of Na⁺ concentration. The interactions between the apoprotein and the lipids are both electrostatic and hydrophobic, but the interaction leading to the conductance increase appears to be mainly hydrophobic. Both the increase in conductance and the current fluctuations remain after extensive washing of the chambers to remove the protein. Furthermore, pronase or glutaraldehyde added to either the cis or trans side of the membrane does not affect the apoprotein-established conductance. However, if the bilayer is formed in the presence of both the apoprotein and pronase or if the apoprotein is treated with pronase prior to its addition to the chamber, no conductance change is observed. The association of the apoprotein with the membrane thus appears to render the protein inaccessible to proteolytic digestion, suggesting that the apoprotein is at least partially imbedded in the membrane interior.     

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.     

Wave-guide spectroscopy and ion transport in planar lipid bilayers

The refractive indices of the bilayer-electrolyte system allow the membrane to operate as a light-guide. This system is then able to monitor, optically, the flow of ions across the bilayer. The light is coupled into and decoupled from a spherically bulged bilayer by means of optical, single mode fibers. The light wave travels along the curved bilayer for several millimeters. This light transmission depends critically on the angle of incidence between the fiber axis and the tangent to the film. Three transmission peaks were observed when the angle of incidence was varied between 0° and 90°. The transmitted light intensity can be modulated by the application of an electric potential upon the bilayer. The center peak, with maximum light transmission, appears at an angle of incidence which is defined by the launching geometry. A quadratic field dependence (independent of the polarity) is observed, which originates from changes in the shape of the torus transition region. The transmission of the satellite peaks, which appear just before and after the central peak, can also be modulated by an external potential. This modulation signal reflects a linear dependence on the polarity of the external voltage. The phase of the modulation signal changes its sign at each satellite peak. It is shown that this modulation signal originates from the bimolecular area of the lipid film. We present evidence that this transmission modulation occurs as a result of ion transport through the lipid film. This provides the basis for the use of wave-guide spectroscopy to investigate membrane ionic fluxes.     

Delta-endotoxins form cation-selective channels in planar lipid bilayers.

Delta-endotoxins CryIA(c) and CryIIIA, two members of a large family of toxic proteins from Bacillus thuringiensis, were each allowed to interact with planar lipid bilayers and were analyzed for their ability to form ion-conducting channels. Both of these toxins made clearly resolved channels in the membranes and exhibited several conductance states, which ranged from 200 pS to about 4000 pS (in 300 mM KCl). The channels formed by both toxins were highly cation-selective, but not ideally so. The permeability ratio of K+ to Cl- was about 25 for both channels. The ability of these proteins to form such channels may account for their toxic action on sensitive cells, and suggests that this family of toxins may act by a common mechanism.     

Toad bladder amiloride-sensitive channels reconstituted into planar lipid bilayers

In the present study we used established methods to obtain apical membrane vesicles from the toad urinary bladder and incorporated these membrane fragments to solvent-free planar lipid bilayer membranes. This resulted in the appearance of a macroscopic conductance highly sensitive to the diuretic amiloride added to the cis side. The blockage is voltage dependent and well described by a model which assumes that the drug binds to sites in the channel lumen. This binding site is localized at about 15% of the electric field across the membrane. The apparent inhibition constant (K(0)) is equal to 0.98 microM. Ca2+, in the micromolar range on the cis side, is a potent blocker of this conductance. The effect of the divalent has a complex voltage dependence and is modulated by pH. At the unitary level we have found two distinct amiloride-blockable channels with conductances of 160 pS (more frequent) and 120 pS. In the absence of the drug the mean open time is around 0.5 sec for both channels and is not dependent on voltage. The channels are cation selective (PNa/PCl = 15) and poorly discriminate between Na+ and K+ (PNa/PK = 2). Amiloride decreases the lifetime in the open state of both channels and also the conductance of the 160-pS channel.     

Orientation of anthracyclines in lipid monolayers and planar asymmetrical bilayers: a surface-enhanced resonance Raman scattering study.

The interaction of anthracyclines (daunorubicin and idarubicin) with monolayers of zwitterionic palmitoyloleoylphosphatidylcholine (POPC) and anionic dipalmitoylphosphatidic acid (POPC-DPPA 80-20 mol%) was studied by surface pressure measurements and compared with previous results obtained with other anthracyclines (pirarubicin and adriamycin). These anthracycline/phospholipid monolayers were next transferred by a Langmuir-Blodgett technique onto planar supports and studied by surface-enhanced resonance Raman scattering (SERRS), which gave information about the orientation of anthracycline in the monolayers. On the whole, the adsorption of anthracyclines in zwitterionic monolayers increases with the anthracycline hydrophobic/hydrophilic balance, which underlines the role of the hydrophobic component of the interaction. On the contrary, the anthracyclines remain adsorbed on the polar headgroups of the phospholipids in the presence of DPPA and form a screen that limits a deeper penetration of other anthracycline molecules. To study by SERRS measurements the crossing of pirarubicin through a phospholipid bilayer used as a membrane model, asymmetrical POPC-DPPA/POPC or POPC/POPC-DPPA bilayers were transferred by the Langmuir-Schäfer method, thanks to a laboratory-built set-up, and put in contact with a pirarubicin aqueous solution. It has been shown that the presence of anionic DPPA in the first monolayer in contact with pirarubicin would limit its crossing. This limiting effet is not observed if the first monolayer is zwitterionic.     

Interactions between a membrane sialoglycoprotein and planar lipid bilayers

Summary Bilayer membranes formed from lipids dissolved in decane were exposed to glycophorin, a sialoglycoprotein which had been extracted from human red cell membranes. The interaction with the bilayer produced an increase in the steady state electrical conductance of the membrane proportional to the amount added. Fluctuations in membrane current when the electrical potential difference was constant were observed concommitantly with this increase in membrane conductance. The minimum size of the fluctuations corresponds to a conductance of 10^–10 mho. The increase in conductance as well as the current fluctuations persisted after extensive washout of the chamber containing the protein (cisside). Subsequent addition of lectins (wheat germ agglutinin and phytohemoagglutinin) to the cis-side produced rupture of the membranes, whilst these hemoagglutinins added to the trans-side failed to produce an effect. Measurements of changes in surface potential using K⁺ nonactin as a probe indicated that glycophorin induces a negative surface charge. At high protein concentrations, the magnitude of the induced surface potential became independent of glycophorin concentration. The maximum number of charges introduced onto the membrane under these conditions was 1.4×10⁵/m². Cis (but not trans)-side addition of neuraminidase abolished these charges, indicating that they can be ascribed to the sialic acid residues that the protein bears. These results suggest that glycophorin incorporates into bilayer membranes with its N-terminal end (where the sialic acid and carbohydrates are located) facing the cis-side. Spectrin reversibly lowered the glycophorin-induced membrane conductance when added to the trans-side. Cis-side additions failed to produce an effect. Trypsin present on the trans-side irreversibly lowered the membrane conductance. These results indicate that parts of the glycophorin molecule, probably the C-terminal end, are accessible to reagents in the solution bathing the trans-side of the membrane. Thus glycophorin spans the planar bilayer in much the same way as it spans the red cell membrane.     

Electrical manipulation of glycan-phosphatidyl inositol-tethered proteins in planar supported bilayers.

Electric fields have been used to manipulate and concentrate glycan-phosphatidyl inositol (GPI)-tethered proteins in planar supported bilayers. Naturally GPI-linked CD48, along with engineered forms of I-Ek and B7-2, in which their transmembrane domains have been genetically replaced with the GPI linkage, were studied. The proteins were labeled with fluorescently tagged antibodies, allowing the electric field-induced behavior to be followed by epifluorescence microscopy. All three protein complexes were observed to migrate toward the cathode with the B7-2 and CD48, each tethered to the membrane by a single GPI linker, moving significantly faster than the I-Ek, which has two GPI linkers. Patterns scratched into the membrane function as barriers to lateral diffusion and were used to isolate the proteins into highly concentrated corrals. All field-induced concentration profiles were completely reversible, indicating that the supported bilayer provides a stable, fluid environment in which GPI-tethered proteins can be manipulated. The ability to electrically control the spatial distribution of membrane-tethered proteins provides new opportunities for the study of biological membranes and the development of membrane-based devices.