Tethered Lipid Membranes Research Articles

Channel activity of a phytotoxin of Clavibacter michiganense ssp. nebraskense in tethered membranes.

Solid-supported membranes immobilized on gold electrodes were used to detect and characterize the spontaneously inserting anion-selective protein channel (Clavibacter anion channel, CAC) present in the culture fluid of Clavibacter michiganense ssp. nebraskense. Three different membrane systems varying in the composition of the first chemisorbed monolayer were investigated by means of impedance spectroscopy. Conductance changes of the immobilized lipid membranes were sensitively detected after adding the culture fluid of the bacteria to the solid-supported membranes, indicating that the relative change in conductance is largest if the lipid layer is attached to the surface via a flexible lipid anchor. Variation in the d.c. potential revealed that CAC exhibits a voltage dependence in these tethered membranes which can be described by an exponential function in accordance with previous results obtained from patchclamp measurements and impedance analysis. The addition of an inhibitor that selectively blocks anion channels abolished the channel conductance almost completely, indicating that the increased conductivity can be attributed to the specific insertion of the CAC. A linear dependence of the channel conductance on the chloride concentration was found, which was modulated by the charges of the second lipid monolayer. The results demonstrate that tethered lipid membranes on gold surfaces in conjunction with impedance spectroscopy allows one to monitor and characterize water-soluble spontaneously inserting channels, providing an effective means to probe for bacterial toxins.

Tethered Bilayer Membranes Containing Ionic Reservoirs: The Interfacial Capacitance

The use of polar linkers to tether lipid bilayer membranes to a gold substrate results in a hydrophilic layer between the membrane and the gold surface. The tethering of lipid bilayer membranes to gold substrates using tetraethylene glycol chains results in a polar layer between the membrane and the gold surface. This region may sequester ions and can act as a reservoir for ions transported across the tethered lipid membrane. In the present article, we report on the electrical properties of this ionic reservoir. In particular, the Stern model of ionic distribution is used to describe the interfacial capacitance. The model combines a surface adsorption layer (Helmholtz model) and a dynamic diffuse layer of ions (Gouy−Chapman model) to describe the interfacial capacitance. This model is used to interpret data from measurements of the interfacial capacitance obtained over a range of ionic species and concentrations. Four analogues of the sulfur−tetraethylene glycol tethers have been investigated. These studies show the effects of varying the structure of the linker group and of introducing a passivation layer adjacent to the gold. Studies were also made of the influence of spacer molecules included to vary the “in-plane” two-dimensional packing. The effect of applying a dc bias potential between an external reference electrode and the gold surface was also studied. These measurements were carried out using ac impedance spectroscopy on bilayers assembled using the method of Cornell et al.6 Most data are successfully modeled as a constant Helmholtz capacitance in series with a diffuse region capacitance that depends on ionic concentration. The dependence on ionic concentration has been modeled by the Gouy−Chapman formalism. At low ionic concentrations (<20 mM), the model becomes inadequate. Deviation from the model also occurs at higher concentrations for more tightly packed membranes, in the absence of tethered spacer molecules. According to the model at very low concentrations of electrolyte, the ionic Debye length intrudes into the hydrocarbon region of the bilayer, violating the Gouy−Chapman assumption of a uniform dielectric medium in the diffuse double layer. The Helmholtz capacitance is insensitive to potential and ionic concentration. This is consistent with Helmholtz capacitance being defined by a hard sphere distance of closest approach of the ions to the gold interface over the range of concentrations studied here. The model suggests that the application of a dc potential alters the permittivity of the diffuse region as a result of water and ions being transported into the reservoir. However, the effective relative permittivity in the reservoir region varies only from 27 to 54, suggesting the reservoir has properties more akin to a dense hydrated gel with restricted ionic mobility than to a bulk electrolyte.

Functionalised interfaces mimicking photosynthesis

Nature has developed different types of photosynthetic molecular assemblies in bacteria, algae and plants. However, in each case the conversion of light into the universal energy currency ATP and the reduction equivalent NADPH is the starting point of anabolic metabolism. We are working on systems where designed interfaces are carrying out these reactions by coupled photoelectrochemistry. In a first step towards mimicking process of ATP regeneration in an artificial membrane we have combined the stability of a solid-supported tethered lipid membrane [1] based on archaebacterial lipids with the light-to-electrochemical energy converting protein, bacteriorhodopsin (bR) [2]. The tethered membrane system is formed onto gold substrates by a gold-sulfur self-assembly process. The membrane spanning lipid contains both a 4nm lipid region as well as a 4nm hydrophilic, oligoethylene glycol region which forms an ionic reservoir between the gold electrode and the lipid membrane Using electrical and electrochemical techniques we have shown that bR can be successfully incorporated and oriented into such tethered lipid membranes. This was seen as a directed proton flux across the lipid membrane into the ionic reservoir in the presence of light. Results obtained were similar to those obtained with Black Lipid Membrane experiments [3]. The current system combines relatively high photoresponses with the stability of tethered archaebacterial lipid membranes. This type of artificial lipid membrane has the potential to act as a matrix for other membrane proteins. An additional advantage of the ionic reservoir is that it allows the incorporation of membrane proteins with large extracellular regions. Incorporation of F0F1-ATPase, which can use the pH gradient to synthesise ATP is one next step in the production of an artificial leaf. The possibility to use these anchor molecules to immobilise redoxcouples like methylviologen and Flavoenzymes [4] to reduce electroenzymatic NADP+ and electron donor, pigment, acceptor multilayer assemblies to supply the electricity are discussed.

S-layer Ultrafiltration Membranes: A New Support for Stabilizing Functionalized Lipid Membranes

We report on a simple method to generate bilayer membranes on a smooth layer of crystalline bacterial cell surface (S-layer) protein lattices, deposited on a microfiltration membrane (MFM), the so-called S-layer ultrafiltration membrane (SUM). Folded bilayers, MFM-, and SUM-supported lipid membranes showed a specific capacitance of 0.6−0.7 μF/cm2. The S-layer produced a stabilizing effect on the lipid membrane; folded and MFM-supported membranes ruptured at the application of a first and a second voltage ramp, respectively, whereas the SUM-supported membranes could withstand multiple voltage ramps. The lifetime of the lipid membranes increased significantly in the order MFM-supported membrane < folded membrane < SUM-supported membrane. By contrast with MFM-supported lipid membranes, successful reconstitution of staphylococcal α-hemolysin (αHL) was observed in both folded and SUM-supported membranes. The unitary conductance of an αHL pore was similar when reconstituted in folded and in SUM-supported lipid membranes. As an alternative to hybrid and tethered lipid membranes, SUM-supported lipid bilayers provide a biomimetic environment for transmembrane proteins.

The peptide-tethered lipid membrane as a biomimetic system to incorporate cytochrome c oxidase in a functionally active form

Peptide-supported lipid bilayers are investigated as a new class of solidsupported membranes tethered to the support by a peptide spacer. They are referred to as peptide tethered lipid membranes (tBLMs), formed by the fusion of liposomes with a thiopeptide-lipid monolayer chemisorbed on a gold support. Peptide tBLMs are designed as a biomimetic system to investigate integral membrane proteins. As an example, cytochrome c oxidase (COX) from bovine heart is incorporated into the preformed peptide tBLM by dilution of the solubilised protein below the critical micellar concentration. The formation of the lipid film as well as the incorporation of the protein were monitored by surface plasmon resonance spectroscopy and surface plasmon fluorescence spectroscopy. COX is activated by adding the reduced form of cytochrome c to the air-saturated buffer solution. Using electrochemical techniques, such as square wave voltammetry (SWV) and chronoamperometry (CA), the direct electron transfer between COX and the gold electrode is observed as well as proton transport from the inside to the outside across the lipid bilayer. Proton transport is then further investigated using impedance spectroscopy, although the electrode is shown to be only partially (70%) covered with a bilayer while defect domains with only a monolayer of peptide or peptide-lipid coexist (approx. 30%). Proton transport carried out by the COX is shown to be voltage dependent. This transport is indicated as a resistance in parallel to the resistance of the lipid film. As a consequence, the total resistance decreases as a function of the concentration of cytochrome c and increases again either by removal of the substrate or by addition of cyanide as an inhibitor of COX. The conductance in the presence of the activated enzyme correlates with the known turnover rate of COX. These experiments demonstrate the possibility to assess the activity of integral membrane proteins incorporated in peptide tBLMs using electrochemical techniques. The system could thus be promising for screening as well as biosensor applications.

Ion-Channel Gating in Transmembrane Receptor Proteins: Functional Activity in Tethered Lipid Membranes.

Through thiolipids a planar lipid bilayer (1) was immobilized on a gold support (2) for use as an electrode. This allows the detection of the ligand-gating function of the natural transmembrane channel protein OmpF (3) reconstituted in the artificial membrane: the binding of a domain (4) of the toxin colicin N, observed by surface plasmon resonance, induces the blocking of the OmpF channel protein, as shown by impedance spectroscopy.