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.     

Temperature dependence of the surface topography in dimyristoylphosphatidylcholine/distearoylphosphatidylcholine multibilayers

Simple lipid binary systems are intensively used to understand the formation of domains in biological membranes. The size of individual domains present in the gel/fluid coexistence region of single supported bilayers, determined by atomic force microscopy (AFM), generally exceeds by two to three orders of magnitude that estimated from multibilayers membranes by indirect spectroscopic methods. In this article, the topography of equimolar dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC) multibilayers, made of two superimposed bilayers supported on mica surface, was studied by AFM in a temperature range from room temperature to 45 degrees C. In the gel/fluid phase coexistence region the size of domains, between approximately 100 nm and a few micrometers, was of the same order for the first bilayer facing the mica and the top bilayer facing the buffer. The gel to fluid phase separation temperature of the first bilayer, however, could be increased by up to 8 degrees C, most likely as a function of the buffer layer thickness that separated it from the mica. Topography of the top bilayer revealed the presence of lipids in ripple phase up to 38-40 degrees C. Above this temperature, a pattern characteristic of the coexistence of fluid and gel domains was observed. These data show that difference in the size of lipid domains given by AFM and spectroscopy can hardly be attributed to the use of multibilayers models in spectroscopy experiments. They also provide a direct evidence for metastable ripple phase transformation into a gel/fluid phase separated structure upon heating.     

Blistering of supported lipid membranes induced by Phospholipase D, as observed by real-time atomic force microscopy

Phospholipase D from Streptomyces chromofuscus (PLDSc) is a soluble enzyme known to be activated by the phosphatidic acid (PA)-calcium complexes. Despite the vast body of literature that has accumulated on this enzyme, the exact mechanism of activation remains poorly understood. In this work, we report the first observation of PLDSc activity in real time and at nanometer resolution using atomic force microscopy (AFM). AFM images of continuous and patchy dipalmitoylphosphatidylcholine (DPPC) bilayers were recorded, prior and after incubation with PLDSc. For continuous bilayers, the enzyme induced important morphological alterations; holes corresponding to the bilayer thickness were created, while an additional elevated phase, about 2.5 nm high, was observed. This bilayer blistering is believed to be due to the production of the negatively charged lipid PA that would cause localized repulsions between the bilayer and the underlying mica surface. By contrast, these elevated domains were not seen on patchy bilayers incubated with the enzyme. Instead, the shapes of DPPC patches were strongly deformed by enzyme activity and evolved into melted morphologies. These results point to the importance of lipid packing on PLD activity and illustrate the potential of AFM for visualizing remodeling enzymatic activities.     

Force measurements on myelin basic protein adsorbed to mica and lipid bilayer surfaces done with the atomic force microscope.

The mechanical and adhesion properties of myelin basic protein (MBP) are important for its function, namely the compaction of the myelin sheath. To get more information about these properties we used atomic force microscopy to study tip-sample interaction of mica and mixed dioleoylphosphatidylserine (DOPS) (20%)/egg phosphatidylcholine (EPC) (80%) lipid bilayer surfaces in the absence and presence of bovine MBP. On mica or DOPS/EPC bilayers a short-range repulsive force (decay length 1.0-1.3 nm) was observed during the approach. The presence of MBP always led to an attractive force between tip and sample. When retracting the tip again, force curves on mica and on lipid layers were different. While attached to the mica surface, the MBP molecules exhibited elastic stretching behavior that agreed with the worm-like chain model, yielding a persistence length of 0.5 +/- 0.25 nm and an average contour length of 53 +/- 19 nm. MBP attached to a lipid bilayer did not show elastic stretching behavior. This shows that the protein adopts a different conformation when in contact with lipids. The lipid bilayer is strongly modified by MBP attachment, indicating formation of MBP-lipid complexes and possibly disruption of the original bilayer structure.     

Following the formation of supported lipid bilayers on mica: a study combining AFM, QCM-D, and ellipsometry

Supported lipid bilayers (SLBs) are popular models of cell membranes with potential biotechnological applications and an understanding of the mechanisms of SLB formation is now emerging. Here we characterize, by combining atomic force microscopy, quartz crystal microbalance with dissipation monitoring, and ellipsometry, the formation of SLBs on mica from sonicated unilamellar vesicles using mixtures of zwitterionic, negatively and positively charged lipids. The results are compared with those we reported previously on silica. As on silica, electrostatic interactions were found to determine the pathway of lipid deposition. However, fundamental differences in the stability of surface-bound vesicles and the mobility of SLB patches were observed, and point out the determining role of the solid support in the SLB-formation process. The presence of calcium was found to have a much more pronounced influence on the lipid deposition process on mica than on silica. Our results indicate a specific calcium-mediated interaction between dioleoylphosphatidylserine molecules and mica. In addition, we show that the use of PLL-g-PEG modified tips considerably improves the AFM imaging of surface-bound vesicles and bilayer patches and evaluate the effects of the AFM tip on the apparent size and shape of these soft structures.     

Blistering of supported lipid membranes induced by Phospholipase D, as observed by real-time atomic force microscopy

Phospholipase D from Streptomyces chromofuscus (PLDSc) is a soluble enzyme known to be activated by the phosphatidic acid (PA)-calcium complexes. Despite the vast body of literature that has accumulated on this enzyme, the exact mechanism of activation remains poorly understood. In this work, we report the first observation of PLDSc activity in real time and at nanometer resolution using atomic force microscopy (AFM). AFM images of continuous and patchy dipalmitoylphosphatidylcholine (DPPC) bilayers were recorded, prior and after incubation with PLDSc. For continuous bilayers, the enzyme induced important morphological alterations; holes corresponding to the bilayer thickness were created, while an additional elevated phase, about 2.5 nm high, was observed. This bilayer blistering is believed to be due to the production of the negatively charged lipid PA that would cause localized repulsions between the bilayer and the underlying mica surface. By contrast, these elevated domains were not seen on patchy bilayers incubated with the enzyme. Instead, the shapes of DPPC patches were strongly deformed by enzyme activity and evolved into melted morphologies. These results point to the importance of lipid packing on PLD activity and illustrate the potential of AFM for visualizing remodeling enzymatic activities.     

Bending elasticities of model membranes: influences of temperature and sterol content

Giant liposomes obtained by electroformation and observed by phase-contrast video microscopy show spontaneous deformations originating from Brownian motion that are characterized, in the case of quasispherical vesicles, by two parameters only, the membrane tension sigma and the bending elasticity k(c). For liposomes containing dimyristoyl phosphatidylcholine (DMPC) or a 10 mol% cholesterol/DMPC mixture, the mechanical property of the membrane, k(c), is shown to be temperature dependent on approaching the main (thermotropic) phase transition temperature T(m). In the case of DMPC/cholesterol bilayers, we also obtained evidence for a relation between the bending elasticity and the corresponding temperature/cholesterol molecular ratio phase diagram. Comparison of DMPC/cholesterol with DMPC/cholesterol sulfate bilayers at 30 degrees C containing 30% sterol ratio shows that k(c) is independent of the surface charge density of the bilayer. Finally, bending elasticities of red blood cell (RBC) total lipid extracts lead to a very low k(c) at 37 degrees C if we refer to DMPC/cholesterol bilayers. At 25 degrees C, the very low bending elasticity of a cholesterol-free RBC lipid extract seems to be related to a phase coexistence, as it can be observed by solid-state (31)P-NMR. At the same temperature, the cholesterol-containing RBC lipid extract membrane shows an increase in the bending constant comparable to the one observed for a high cholesterol ratio in DMPC membranes.     

Interactions of the local anesthetic tetracaine with membranes containing phosphatidylcholine and cholesterol: a 2H NMR study

The interactions of the local anesthetic tetracaine with multilamellar dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol have been investigated by deuterium nuclear magnetic resonance of specifically deuteriated tetracaines, DMPC and cholesterol. Experiments were performed at pH 5.5, when the anesthetic is primarily charged, and at pH 9.5, when it is primarily uncharged. The partition coefficients of the anesthetic in the membrane have been measured at both pH values for phosphatidylcholine bilayers with and without cholesterol. The higher partition coefficients obtained at pH 9.5 reflect the hydrophobic interactions between the uncharged form of the anesthetic and the hydrocarbon region of the bilayer. The lower partition coefficients for the DMPC/cholesterol system at both pH values suggest that cholesterol, which increases the order of the lipid chains, decreases the solubility of tetracaine into the bilayer. For phosphatidylcholine bilayers, it has been proposed [Boulanger, Y., Schreier, S., & Smith, I. C. P. (1981) Biochemistry 20, 6824-6830] that the charged tetracaine at low pH is located mostly at the phospholipid headgroup level while the uncharged tetracaine intercalates more deeply into the bilayer. The present study suggests that the location of tetracaine in the cholesterol-containing system is different from that in pure phosphatidylcholine bilayers: the anesthetic sits higher in the membrane. An increase in temperature results in a deeper penetration of the anesthetic into the bilayer. Moreover, the incorporation of the anesthetic into DMPC bilayers with or without cholesterol results in a reduction of the lipid order parameters both in the plateau and in the tail regions of the acyl chains, this effect being greater with the charged form of the anesthetic.     

Galactosylceramide domain microstructure: impact of cholesterol and nucleation/growth conditions

Galactosylceramide (GalCer), a glycosphingolipid, is believed to exist in the extracellular leaflet of cell membranes in nanometer-sized domains or rafts. The local clustering of GalCer within rafts is thought to facilitate the initial adhesion of certain viruses, including HIV-1, and bacteria to cells through multivalent interactions between receptor proteins (gp120 for HIV-1) and GalCer. Here we use atomic force microscopy (AFM) to study the effects of cholesterol on solid-phase GalCer domain microstructure and miscibility with a fluid lipid 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) in supported lipid bilayers. Using "slow-cooled vesicle fusion" to prepare the supported lipid bilayers, we were able to overcome the nonequilibrium effects of the substrate (verified by comparison to results for giant unilamellar vesicles) and accurately quantify the dramatic effect of cholesterol on the GalCer domain surface area/perimeter ratio (A(D)/P) and DLPC-GalCer miscibility. We compare these results to a supported lipid bilayer system in which the bilayer is rapidly cooled (nonequilibrium conditions), "quenched vesicle fusion", and find that the microstructures are remarkably similar above a cholesterol mol fraction of approximately 0.06. We determined that GalCer domains were contained in one leaflet distal to the mica substrate through qualitative binding experiments with Trichosanthes kirilowii agglutinin (TKA), a galactose-specific lectin, and AFM of Langmuir-Blodgett deposited GalCer/DLPC supported lipid bilayers. In addition, GalCer domains in bilayers containing cholesterol rearranged upon tip-sample contact. Our results further serve to clarify why discrepancies exist between different model membrane systems and between model membranes and cell membranes. In addition, these results offer new insight into the effect of cholesterol and surrounding lipid on domain microstructure and behavior. Finally, our observations may be pertinent to cell membrane structure, dynamics, and HIV infection.     

Interaction of cationic surfactants with DPPC membranes: effect of a novel Nα-benzoylated arginine-based compound

Cationic amino acid-based surfactants are known to interact with the lipid bilayer of microorganism resulting in cell death through a disruption of the membrane topology. To elucidate the interaction of a cationic surfactant synthesized in our lab, investigations involving N^α-benzoyl-arginine decyl amide (Bz-Arg-NHC₁₀), and model membranes composed by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were done. Bz-Arg-NHC₁₀was able to penetrate into DPPC monolayers up to a critical pressure of 59.6 mN m⁻¹. Differential scanning calorimetry revealed that as the concentration of Bz-Arg-NHC₁₀ increased, the main transition temperature of DPPC slightly decreased. Atomic force microscopy (AFM) in situ experiments performed on supported DPPC bilayers on mica allowed monitoring the changes induced by Bz-Arg-NHC₁₀. DPPC bilayer patches were partially removed, mainly in borders and bilayer defects for 50 µM Bz-Arg-NHC₁₀ solution. Increasing the concentration to 100 µM resulted in a complete depletion of the supported bilayers. Surface plasmon resonance (SPR) experiments, carried out with fully DPPC bilayers covered chips, showed a net increase of the SPR signal, which can be explained by Bz-Arg-NHC₁₀ adsorption. When patchy DPPC bilayers were formed on the substrate, a SPR signal net decrease was obtained, which is consistent with the phospholipids’ removal observed in the AFM images. The results obtained suggest that the presence of the benzoyl group attached to the polar head of our compound would be the responsible of the increased antimicrobial activity against gram-negative bacteria when compared with other arginine-based surfactants.

     

Supported planar bilayer formation by vesicle fusion: the interaction of phospholipid vesicles with surfaces and the effect of gramicidin on bilayer properties using atomic force microscopy

We have used magnetic alternating current mode atomic force microscopy (MAC-AFM) to investigate the formation of supported phospholipid bilayers (SPB) by the method of vesicle fusion. The systems studied were dioleoylphosphatidylcholine (DOPC) on mica and mica modified with 3-aminopropyl-triethoxy-silane (APTES), and DOPC vesicles with gramicidin incorporated on mica and APTES-modified mica. The AFM images reveal three stages of bilayer formation: localized disklike features that are single bilayer footprints of the vesicles, partial continuous coverage, and finally complete bilayer formation. The mechanism of supported phospholipid bilayers formation is the fusion of proximal vesicles, rather than surface disk migration. This mechanism does not appear to be affected by incorporation of gramicidin or by surface modification. Once formed, the bilayer develops circular defects one bilayer deep. These defects grow in size and number until a dynamic equilibrium is reached.     

Polymer-cushioned bilayers. II. An investigation of interaction forces and fusion using the surface forces apparatus.

We have created phospholipid bilayers supported on soft polymer "cushions" which act as deformable substrates (see accompanying paper, Wong, J. Y., J. Majewski, M. Seitz, C. K. Park, J. N. Israelachvili, and G. S. Smith. 1999. Biophys. J. 77:1445-1457). In contrast to "solid-supported" membranes, such "soft-supported" membranes can exhibit more natural (higher) fluidity. Our bilayer system was constructed by adsorption of small unilamellar dimyristoylphosphatidylcholine (DMPC) vesicles onto polyethylenimine (PEI)-supported Langmuir-Blodgett lipid monolayers on mica. We used the surface forces apparatus (SFA) to investigate the long-range forces, adhesion, and fusion of two DMPC bilayers both above and below their main transition temperature (T(m) approximately 24 degrees C). Above T(m), hemi-fusion activation pressures of apposing bilayers were considerably smaller than for solid-supported bilayers, e.g., directly supported on mica. After separation, the bilayers naturally re-formed after short healing times. Also, for the first time, complete fusion of two fluid (liquid crystalline) phospholipid bilayers was observed in the SFA. Below T(m) (gel state), very high pressures were needed for hemi-fusion and the healing process became very slow. The presence of the polymer cushion significantly alters the interaction potential, e.g., long-range forces as well as fusion pressures, when compared to solid-supported systems. These fluid model membranes should allow the future study of integral membrane proteins under more physiological conditions.     

The membrane insertion of helical antimicrobial peptides from the N-terminus of Helicobacter pylori ribosomal protein L1

The interaction of two helical antimicrobial peptides, HPA3 and HPA3P with planar supported lipid membranes was quantitatively analysed using two complementary optical biosensors. The peptides are analogues of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1). The binding of these two peptide analogues to zwitterionic dimyristoyl-phosphatidylcholine (DMPC) and negatively charged membranes composed of DMPC/dimyristoylphosphatidylglycerol (DMPG) (4:1) was determined using surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). Using SPR analysis, it was shown that the proline substitution in HPA3P resulted in much lower binding for both zwitterionic and anionic membranes than HPA3. Structural changes in the planar DMPC and DMPC/DMPG (4:1) bilayers induced by the binding of both Hp(2-20) analogues were then resolved in real-time with DPI. The overall process of peptide-induced changes in membrane structure was analysed by the real-time changes in bound peptide mass as a function of bilayer birefringence. The insertion of both HPA3 and HPA3P into the supported lipid bilayers resulted in a decrease in birefringence with increasing amounts of bound peptide which reflects a decrease in the order of the bilayer. The binding of HPA3 to each membrane was associated with a higher level of bound peptide and greater membrane lipid disordering and a faster and higher degree of insertion into the membrane than HPA3P. Furthermore, the binding of both HPA3 and HPA3P to negatively charged DMPC/DMPG bilayers also leads to a greater disruption of the lipid ordering. These results demonstrate the geometrical changes in the membrane upon peptide insertion and the extent of membrane structural changes can be obtained quantitatively. Moreover, monitoring the effect of peptides on a structurally characterised bilayer has provided further insight into the role of membrane structure changes in the molecular basis of peptide selectivity and activity and may assist in defining the mode of antimicrobial action.     

Structural effects of the antimicrobial peptide maculatin 1.1 on supported lipid bilayers

The interactions of the antimicrobial peptide maculatin 1.1 (GLFGVLAKVAAHVVPAIAEHF-NH₂) with model phospholipid membranes were studied by use of dual polarisation interferometry and neutron reflectometry and dimyristoylphosphatidylcholine (DMPC) and mixed DMPC–dimyristoylphosphatidylglycerol (DMPG)-supported lipid bilayers chosen to mimic eukaryotic and prokaryotic membranes, respectively. In DMPC bilayers concentration-dependent binding and increasing perturbation of bilayer order by maculatin were observed. By contrast, in mixed DMPC–DMPG bilayers, maculatin interacted more strongly and in a concentration-dependent manner with retention of bilayer lipid order and structure, consistent with pore formation. These results emphasise the importance of membrane charge in mediating antimicrobial peptide activity and emphasise the importance of using complementary methods of analysis in probing the mode of action of antimicrobial peptides.     

Membrane topology of a 14-mer model amphipathic peptide: a solid-state NMR spectroscopy study

We have investigated the interaction between a synthetic amphipathic 14-mer peptide and model membranes by solid-state NMR. The 14-mer peptide is composed of leucines and phenylalanines modified by the addition of crown ethers and forms a helical amphipathic structure in solution and bound to lipid membranes. To shed light on its membrane topology, 31P, 2H, 15N solid-state NMR experiments have been performed on the 14-mer peptide in interaction with mechanically oriented bilayers of dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), and dipalmitoylphosphatidylcholine (DPPC). The 31P, 2H, and 15N NMR results indicate that the 14-mer peptide remains at the surface of the DLPC, DMPC, and DPPC bilayers stacked between glass plates and perturbs the lipid orientation relative to the magnetic field direction. Its membrane topology is similar in DLPC and DMPC bilayers, whereas the peptide seems to be more deeply inserted in DPPC bilayers, as revealed by the greater orientational and motional disorder of the DPPC lipid headgroup and acyl chains. 15N{31P} rotational echo double resonance experiments have also been used to measure the intermolecular dipole-dipole interaction between the 14-mer peptide and the phospholipid headgroup of DMPC multilamellar vesicles, and the results indicate that the 14-mer peptide is in contact with the polar region of the DMPC lipids. On the basis of these studies, the mechanism of membrane perturbation of the 14-mer peptide is associated to the induction of a positive curvature strain induced by the peptide lying on the bilayer surface and seems to be independent of the bilayer hydrophobic thickness.     

Peroxidase immobilized on phospholipid bilayers supported on au (111) by DTT self‐assembled monolayers: Application to dopamine determination

In this work, horseradish peroxidase (HRP) was immobilized on dimyristoylphosphatidylcholine (DMPC) bilayers supported on Au (111) by dithiotreitol (DTT) self‐assembled monolayers and used as a nanostructured electrochemical biosensor to dopamine determination. The morphology of the phospholipid bilayers and the immobilization of HRP to these layers were characterized by atomic force microscopy (AFM). Square‐wave voltammetry (SWV) experiments were done to investigate the performance of the HRP‐modified electrode. The AFM images indicate that the enzyme is adsorbed at the external layer of the lipid bilayer and, although the electrical charges on the surface were not measured, the enzyme and phospholipids surface interaction occurs probably by electrostatic forces due to the pH used in the experiments. Interestingly, the present system can be used as one‐shot sensor for the rapid detection of dopamine. The analytical performance of this system was linear for dopamine concentrations from 3.3 × 10^?5 to 1.3 × 10^?3 mol L^?1 (r = 0.9997) with a detection limit of 2.0 × 10^?6 mol L^?1. Our results indicate that the use of HRP‐DMPC bilayer system may be useful not only in developing new nanostructured materials for technological purposes, but could be very useful in fundamental studies to investigate the interactions between different micro‐and macromolecules, even with soluble proteins, and lipid membranes. Biotechnol. Bioeng. 2013; 110: 374–382. © 2012 Wiley Periodicals, Inc.     

Growth of cationic lipid toward bilayer lipid membrane by solution spreading: scanning probe microscopy study

The growth of cationic lipid dioctadecyldimethylammonium bromide (DODAB) toward bilayer lipid membrane (BLM) by solution spreading on cleaved mica surface was studied by atomic force microscopy (AFM). Bilayer of DODAB was formed by exposing mica to a solution of DODAB in chloroform and subsequently immersing into potassium chloride solution for film developing. AFM studies showed that at the initial stage of the growth, the adsorbed molecules exhibited the small fractal-like aggregates. These aggregates grew up and expanded laterally into larger patches with time and experienced from monolayer to bilayer, finally a close-packed bilayer film (5.4+/-0.2 nm) was approached. AFM results of the film growth process indicated a growth mechanism of nucleation, growth and coalescence of dense submonolayer, it revealed the direct information about the film morphology and confirmed that solution spreading was an effective technique to prepare a cationic bilayer in a short time.     

Visualization of membrane RNAs

Using fluorescence microscopy, we show that previously isolated membrane-binding RNAs coat artificial phospholipid membranes relatively uniformly, except for a frequent tendency to concentrate at bends, membrane junctions, and other unusual sites. Membrane RNAs can also be visualized as single molecules or isolated complexes by atomic force microscopy (AFM) of free RNAs on mica. Finally, RNAs can be seen within membranes by AFM of RNA-liposomes immobilized on hydrophobic mica surfaces. Monomer RNAs appear globular, as expected for small RNAs. When mixed under conditions in which RNAs bind bilayers, RNA 9 and RNA 10 combine to yield about 80% of RNAs as mainly linear oligomers of approximately 2-8 molecules. Once inserted in membranes, the RNAs oligomerize further, yielding larger, irregular ropelike structures that prefer the edges of altered lipid patches. These properties can be interpreted in terms of RNA-RNA loop interactions, and the RNA effects on membranes can be explained in terms of an RNA preference for irregular lipid conformations. The RNA-bilayer system poses new opportunities for combining the properties of membranes and RNA in contemporary cells, and also in the ribocytes of an RNA world.     

Structure of supported bilayers composed of lipopolysaccharides and bacterial phospholipids: raft formation and implications for bacterial resistance

Lipopolysaccharide (LPS), the major lipid on the surface of Gram-negative bacteria, plays a key role in bacterial resistance to hydrophobic antibiotics and antimicrobial peptides. Using atomic force microscopy (AFM) we characterized supported bilayers composed of LPSs from two bacterial chemotypes with different sensitivities to such antibiotics and peptides. Rd LPS, from more sensitive "deep rough" mutants, contains only an inner saccharide core, whereas Ra LPS, from "rough" mutants, contains a longer polysaccharide region. A vesicle fusion technique was used to deposit LPS onto either freshly cleaved mica or polyethylenimine-coated mica substrates. The thickness of the supported bilayers measured with contact-mode AFM was 7 nm for Rd LPS and 9 nm for Ra LPS, consistent with previous x-ray diffraction measurements. In water the Ra LPS bilayer surface was more disordered than Rd LPS bilayers, likely due to the greater volume occupied by the longer Ra LPS polysaccharide region. Since deep rough mutants contain bacterial phospholipid (BPL) as well as LPS on their surfaces, we also investigated the organization of Rd LPS/BPL bilayers. Differential scanning calorimetry and x-ray diffraction indicated that incorporation of BPL reduced the phase transition temperature, enthalpy, and average bilayer thickness of Rd LPS. For Rd LPS/BPL mixtures, AFM showed irregularly shaped regions thinner than Rd LPS bilayers by 2 nm (the difference in thickness between Rd LPS and BPL bilayers), whose area increased with increasing BPL concentration. We argue that the increased permeability of deep rough mutants is due to structural modifications caused by BPL to the LPS membrane, in LPS hydrocarbon chain packing and in the formation of BPL-enriched microdomains.     

Transfer on hydrophobic substrates and AFM imaging of membrane proteins reconstituted in planar lipid bilayers

The lipid-layer technique allows reconstituting transmembrane proteins at a high density in microns size planar membranes and suspended to a lipid monolayer at the air/water interface. In this paper, we transferred these membranes onto two hydrophobic substrates for further structural analysis of reconstituted proteins by Atomic Force Microscopy (AFM). We used a mica sheet covered by a lipid monolayer or a sheet of highly oriented pyrolytic graphite (HOPG) to trap the lipid monolayer at the interface and the suspended membranes. In both cases, we succeeded in the transfer of large membrane patches containing densely packed or 2D-crystallized proteins. As a proof of concept, we transferred and imaged the soluble Shiga toxin bound to its lipid ligand and the ATP-binding cassette (ABC) transporter BmrA reconstituted into a planar bilayer. AFM imaging with a lateral resolution in the nanometer range was achieved. Potential applications of this technique in structural biology and nanobiotechnology are discussed.