Supported Planar Bilayers Research Articles

Interfacial Membrane Effects of Fluoroquinolones as Revealed by a Combination of Fluorescence Binding Experiments and Atomic Force Microscopy Observations

6-Fluoroquinolones are useful antimicrobial agents against gram-positive and gram-negative bacteria and some mycobacterial species as well. Although the diffusion through porins in gram-negative bacteria is well established, other mechanisms such as the hydrophobic pathway through the apolar regions of the bilayer and the self-promoted pathway appear to be relevant or concomitant with the hydrophilic pathway in many cases. This article discusses the interaction of ciprofloxacin (CPX) and two new synthesized compounds (M3CPX and M4CPX)-with a methyl group attached at the N3 and N4 positions of the piperazynil ring of the CPX-with liposomes and supported planar bilayers (SPBs) of Escherichia coli. Binding experiments using ANS revealed that the three compounds interact electrostatically with the bilayer. The variations in the electrostatic surface potential, which is always positive, were higher for M3CPX than for CPX or M4CPX. Related to that, the SPBs of E. coli were more affected by M3CPX than by the other two compounds, as judged by the analysis of the atomic force microcopy (AFM) images. The in situ injection of the three 6-fluoroquinolones (6-FQs) induced different changes in height, roughness (Ra), and area covered by the SPBs.

Supported planar bilayers from hexagonal phases

In this work the presence of inverted hexagonal phases H II of 1-palmitoy-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE) and cardiolipin (CL) (0.8:0.2, mol/mol) in the presence of Ca 2+ were observed via 31P-NMR spectroscopy. When suspensions of the same composition were extended onto mica, H II phases transformed into structures which features are those of supported planar bilayers (SPBs). When characterized by atomic force microscopy (AFM), the SPBs revealed the existence of two laterally segregated domains (the interdomain height being ∼ 1 nm). Cytochrome c (cyt c), which binds preferentially to acidic phospholipids like CL, was used to demonstrate the nature of the domains. We used 1-anilinonaphtalen-8-sulfonate (ANS) to demonstrate that in the presence of cyt c, the fluorescence of ANS decreased significantly in lamellar phases. Conversely, the ANS binding to H II phases was negligible. When cyt c was injected into AFM fluid imaging cells, where SPBs of POPE:CL had previously formed poorly defined structures, protein aggregates (∼ 100 nm diameter) were ostensibly observed only on the upper domains, which suggests not only that they are mainly formed by CL, but also provides evidence of bilayer formation from H II phases. Furthermore, a model for the nanostructure of the SPBs is herein proposed.

Thermodynamic and structural study of the main phospholipid components comprising the mitochondrial inner membrane

Cardiolipin (CL) is a phospholipid found in the energy-transducing membranes of bacteria and mitochondria and it is thought to be involved in relevant biological processes as apoptosis. In this work, the mixing properties of CL and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocoline (POPC) and 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphoethanolamine (POPE) at the air–water interface, have been examined using the thermodynamic framework analysis of compression isotherms. Accordingly, the values of the Gibbs energy of mixing, the more stable monolayers assayed were: POPC:CL (0.6:0.4, mol:mol) and POPE:CL (0.8:0.2, mol:mol). The results reflect that attractive forces are the greatest contributors to the total interaction in these compositions. Supported planar bilayers (SPBs) with such compositions were examined using atomic force microscopy (AFM) at different temperatures. With the POPC:CL mixture, rounded and featureless SPBs were obtained at 4 °C and 24 °C. In contrast, the extension of the POPE:CL mixture revealed the existence of different lipid domains at 24 °C and 37 °C. Three lipid domains coexisted which can be distinguished by measuring the step height difference between the uncovered mica and the bilayer. While the low and intermediate domains were temperature dependent, the high domain was composition dependent. When cytochrome c (cyt c) was injected into the fluid cell, the protein showed a preferential adsorption onto the high domain of the POPC:CL. These results suggest that the high domain is mainly formed by CL.

S1e2-0 New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-0 New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-5 Immunosensors based on Tethered Lipid Membranes(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-5 Immunosensors based on Tethered Lipid Membranes(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-8 Micropatterned composite membranes of polymerized and fluid lipid bilayers as a versatile model cellular membrane(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-8 Micropatterned composite membranes of polymerized and fluid lipid bilayers as a versatile model cellular membrane(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-2 Direct observation of process of various transformations that induced into giant liposome(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-2 Direct observation of process of various transformations that induced into giant liposome(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-7 Optical and electrical detection of single drug bindings to a single ion-channel protein(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-7 Optical and electrical detection of single drug bindings to a single ion-channel protein(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-6 Fabrication of Ion-channel Biosensor and Interface Control(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-6 Fabrication of Ion-channel Biosensor and Interface Control(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-4 Toward the creation of de novo cell : Modeling of cellular structure and function(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-4 Toward the creation of de novo cell : Modeling of cellular structure and function(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-3 Population analysis of liposomes with protein synthesis and a cascading genetic network(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

S1e2-3 Population analysis of liposomes with protein synthesis and a cascading genetic network(S1-e2: "New Biomembrane Model Systems, Giant Liposomes and Supported Planar Bilayers, for Probing Biomembrane Structure and Function, and Creation of De Novo Functional Membrane System",Symposia,Abstract,Meeting Program of EABS & BSJ 2006)

Induced structural changes of a supported planar bilayer after exposure to halothane — A real-time atomic force microscopy study

Atomic force microscopy (AFM) was used to study the effect of the general anesthetic halothane on a supported DOPC phospholipid bilayer, under conditions of high anaesthetic loading. The presence of the anaesthetic in the lipid-induced restructuring of the supported bilayer, erosion of the bilayer from the mica surface, and the formation of lipid aggregates. Addition of halothane to lipid vesicles prior to adsorption on the mica surface inhibited the lysis of adsorbed vesicles and thus no planar bilayer formation was observed.Key words: lipid bilayer restructuring, atomic force microscopy, halothane, anesthesis.

Effects of Lactose Permease on the Phospholipid Environment in Which It Is Reconstituted: A Fluorescence and Atomic Force Microscopy Study

The membrane transport protein lactose permease (LacY), a member of the major facilitator superfamily containing 12 membrane-spanning segments connected by hydrophilic loops, was reconstituted in liposomes whose composition was 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol in a 3:1 molar ratio. The structural order of the lipid membranes, in the presence and absence of LacY, was assessed using steady-state fluorescence anisotropy. The features of the anisotropy curves obtained with 1,6-phenyl-1,3,5-hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate suggest a surface effect of LacY on the membranes. Atomic force microscopy imaging of supported planar bilayers (SPBs) deposited onto mica was used to examine the effect of LacY on the nanostructure of the phospholipid matrix. Two separated domains were observed in SPBs formed from pure phospholipid mixture. Protein assemblies segregated from the rest of the matrix were observed after the extension of proteoliposomes. The effect of the protein on the electrostatic surface potential of the bilayer was also examined using a fluorescent pH indicator, 4-heptadecyl-7-hydroxycoumarin. Changes in surface potential were enhanced in the presence of the substrate (i.e., lactose). Taken together the results indicate that LacY is segregated into the phospholipid matrix and has moderate effects on the acyl chain order of the bilayers. The changes in surface electrical properties of the bilayers suggest a role for the phospholipid headgroups in proton transfer to the amino acids involved in substrate translocation.

Investigating porcine pancreatic phospholipase A2 action on vesicles and supported planar bilayers using a quartz crystal microbalance with dissipation.

We present an investigation of the activity of porcine pancreatic phospholipase A2 towards phospholipids. The phospholipids are presented in three different ways, namely as tethered vesicles, intact surface-bound vesicles, and supported planar bilayers (SPBs). The process is followed using a quartz crystal microbalance which measures both the frequency shift and the energy dissipation factor. This technique is very sensitive not only to the mass of the material deposited on the crystal, but also to its viscoelasticity. The breakdown of the phospholipid vesicles and bilayers consequently gives rise to very large signal changes. Enzyme binding is separated from vesicle hydrolysis using nonhydrolyzable ether lipids. Intact and tethered vesicles give rise to the same profile, indicating that direct immobilization of the vesicles does not affect hydrolysis significantly. The data fit well to a Voight-based model describing the change in film structure with time. Initial enzyme binding to intact vesicles is accompanied by a significant increase in layer thickness as well as a decrease in viscosity and shear modulus. This effect, which is less pronounced in SPBs, is probably mainly due to the accumulation of hydrolysis products in the vesicle prior to rupture of the vesicles and release of bound water, since it disappears when lysolipid is included in the vesicles prior to hydrolysis.

Deposition of phospholipid layers on SiO2 surface modified by alkyl-SAM islands

Formation of the supported planar bilayer of dipalmitoylphosphatidylcholine (DPPC) on SiO2 surfaces modified with the self-assembled monolayer (SAM) of octadecyltrichlorosilane (OTS) has been investigated by atomic force microscopy (AFM). DPPC was deposited by the fusion of vesicles on SiO2 surfaces with OTS-SAM islands of different sizes and densities. The DPPC bilayer membrane formed self-organizingly on the SiO2 surface with small and sparse OTS islands, while did not when the OTS islands were larger and denser. The relative size between the vesicles and the SiO2 regions is the critical factor for the formation of the DPPC bilayer membrane.

Revisiting lipid – general anesthetic interactions (I): Thinned domain formation in supported planar bilayers induced by halothane and ethanol

A long-standing question in anesthesia is that of the molecular mechanism. Do anesthetics target proteins or change membrane properties or both? We used temperature-dependent magnetic A/C mode atomic force microscopy (AFM) to study interaction of the volatile anesthetics halothane and ethanol with model membranes made from supported planar bilayers (SPBs) of 1,2-dioleoyl-sn-3-glycero-3-phosphocholine (DOPC), dioleoyltrimethylammonium propane (DOTAP), or 1,2-dipalmitoyl-sn-3-glycero-3-phosphocholine (DPPC). We found that the incorporation of halothane or ethanol induces structural changes in the bilayer. These compounds cause thickness reduction in Lα bilayers (either globally or in domains) and the formation of domains with reduced thickness in Lβ phase bilayers. We propose that an anesthetic-induced increased area per lipid drives local chain disorder, thus promoting local phase change. The characteristics of SPBs with halothane or ethanol incorporated were compared with characteristics of the Lα and Lβ phases of anesthetic-free SPBs.Key words: atomic force microscopy, anesthesia, lipid bilayer domains, phase transition

Imaging single membrane fusion events mediated by SNARE proteins.

Using total internal reflection fluorescence microscopy, we have developed an assay to monitor individual fusion events between proteoliposomes containing vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and a supported planar bilayer containing cognate target SNAREs. Approach, docking, and fusion of individual vesicles to the target membrane were quantified by delivery and subsequent lateral spread of fluorescent phospholipids from the vesicle membrane into the target bilayer. Fusion probability was increased by raising divalent cations (Ca2+ and Mg2+). Fusion of individual vesicles initiated in <100 ms after the rise of Ca2+ and membrane mixing was complete in 300 ms. Removal of the N-terminal H(abc) domain of syntaxin 1A increased fusion probability >30-fold compared to the full-length protein, but even in the absence of the H(abc) domain, vesicle fusion was still enhanced in response to Ca2+ increase. Our observations establish that the SNARE core complex is sufficient to fuse two opposing membrane bilayers at a speed commensurate with most membrane fusion processes in cells. This real-time analysis of single vesicle fusion opens the door to mechanistic studies of how SNARE and accessory proteins regulate fusion processes in vivo.

Single molecule imaging of supported planar lipid bilayer—reconstituted human insulin receptors by in situ scanning probe microscopy

A 480-kDa disulfide-linked heterodimer single-pass transmembrane protein, the insulin receptor, is autophosphorylated upon insulin binding to its extracellular domain. Remarkably, the structural basis for this activation process remained largely unknown until the recent cryoelectron microscopy studies of the insulin–insulin receptor complex by Luo et al. [Science 285 (1999) 1077]. We report here the results of an in situ study by high-resolution scanning probe microscopy of the full-length insulin receptor reconstituted within supported planar lipid bilayers. Our preliminary studies confirm that (1) the intact receptor can be reconstituted constitutively within a lipid vesicle and (2) fusion of the receptor-containing vesicles to mica resulted in the formation of molecular flat 5.5-nm-thick supported planar bilayers populated by two populations of protrusions, the shape and size of which are consistent with those of the insulin receptor's intra- and extracellular domains as modeled by the cryo-EM data of Ottensmeyer et al. [Biochemistry 39 (2000) 12103]. These results establish a framework for real-time studies of insulin–insulin receptor binding by in situ SPM and single molecule force spectroscopy.

Bioadhesion of supramolecular structures at supported planar bilayers as studied by the quartz crystal microbalance.

A quartz crystal microbalance (QCM) was used to study the adhesion behavior of supramolecular aggregates at supported planar bilayers (SPBs). The QCM technique is a suitable method to detect the adsorption of biomolecules at the quartz surface owing to its sensitivity for changes in mass and viscoelastic properties. To simulate biomembranes, the quartz plates were coated with highly ordered lipid films. Therefore, a combination of self-assembled monolayers and Langmuir-Blodgett films was used. Firstly, the adsorption of liposomes coupled with the lectin concanavalin A was investigated at glycolipid-containing model membranes. Using different carbohydrates, it was possible to determine specific and nonspecific parts of the interactions. The adhesion occurred owing to specific lectin-carbohydrate interactions (about 20%) and to nonspecific interactions (about 80%). The composition of the liposomes was changed to simulate the structure of a native biomembrane consisting of the glycocalix, the lipid-protein bilayer, and the cytoskeleton. An artificial glycocalix was created by incorporating poly(ethylene glycol) into the liposomes. Liposomes which were intravesicular polymerized with polyacrylamide or polyacrylcholate simulated the cytoskeleton. It was determined that the modified liposomes had significant lower interactions with SPBs. The adsorption was reduced by approximately 80% compared to unmodified liposomes. Secondly, a model was developed for the detection of interactions between simple or mixed bile salt micelles and model membranes. It was found that simple bile salts did not adsorb at model membranes. Binary systems consisting of bile salt and phospholipid induced only small interactions. On the other hand, ternary systems consisting of bile salt, phospholipid, and fatty acid showed strong interactions. A dependence on the chain length of the fatty acid was observed. Thirdly, the interaction between ganglioside-containing model membranes and cholera toxin (beta-subunit) was investigated. Different ganglioside fractions showed varying adsorption in the following sequence: GM1 > GD1a > GD1b > GT1b.

Formation of domains of cationic or anionic lipids in binary lipid mixtures increases the electrostatic coupling strength of water-soluble proteins to supported bilayers.

The electrostatic binding strength of water-soluble proteins having either an excess positive (cytochrome c) or negative (beta-lactoglobulin) electric charge to oppositely charged supported planar bilayers (SPBs) was studied as a function of the bilayer phase state (fluid or gel phase) by IR-ATR spectroscopy. The bilayer consisted of mixtures of zwitterionic DEPC with either cationic DMTAP or anionic DMPG. We observed drastic differences in the binding strength of both proteins for the two bilayer phase states, with the gel phase exhibiting a higher binding strength than the fluid phase, under conditions where the two lipid components had different hydrophobic chain lengths resulting in a nonideal mixing behavior. In addition, for beta-lactoglobulin we observed a strong binding to a gel phase SPB comprising DEPC/DMTAP, while raising the temperature of the SPB above the chain melting transition temperature of the mixture resulted in a complete unbinding of the protein. In contrast, for DMPC/DMTAP having the same cationic charge content but no hydrophobic chain mismatch, no phase-dependent coupling strength of the protein to the SPB was observed. Our results suggest that the formation of charge-enriched domains by partial demixing of the bilayer lipids at the transition to the gel state is crucial for modulation of the protein binding strength to the SPB, while the intrinsic charge of the solid support surface is of minor importance.