Renate Naumann

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

Abstract 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.

Proton transport through a peptide-tethered bilayer lipid membrane by the H+-ATP synthase from chloroplasts measured by impedance spectroscopy

A lipid membrane was tethered to a gold film by a peptide spacer molecule terminated by a sulfhydryl group. Membranes were formed by fusion of liposomes prepared from egg phosphatidylcholine on self assembled monolayers of the thiolipopeptide Myr-Lys(Myr)-Ser-Ser-Pro-Ala-Ser-Ser-Ala-Ala-Ser-Ala-Cys-amide mixed with mercaptoethanol as a diluent molecule or lateral spacer. These mixed films, although not representing a perfect lipid bilayer, have been shown to retain the activity of incorporated H(+)-ATP synthases from chloroplasts in contrast to films prepared from the pure thiolipopeptide. The activity of the protein was demonstrated by impedance spectroscopy. The resistance decreased due to proton transport across the lipid film, which occurs as a consequence of adenosine triphosphate (ATP) hydrolysis. Several effects previously determined from kinetic measurements of the enzyme reconstituted in liposomes such as saturation with respect to the substrate (ATP), inhibition by venturicidin, activation by a positive potential pulse and increase of the proton current as a function of increasingly negative potentials have been confirmed also for this tethered membrane system. Changes in the impedance spectra due to the addition of ATP were fully reversible.

Incorporation of the acetylcholine receptor dimer from Torpedo californica in a peptide supported lipid membrane investigated by surface plasmon and fluorescence spectroscopy

The dimer species (M(r) 580,000) of the nicotinic acetylcholine receptor, isolated from the electric organ of Torpedo californica, was incorporated into a thiopeptide supported lipid bilayer. The incorporation was achieved by fusion of liposomes with reconstituted receptor onto a gold-supported thiopeptide lipid monolayer. Surface plasmon resonance spectroscopy (SPS) was used to monitor in real time the fusion process as well as the specific binding of the antagonist alpha-bungarotoxin. A recently developed extension of SPS offering enhanced sensitivity and specificity, surface plasmon fluorescence spectroscopy (SPFS), was then used to monitor subsequent binding of the monoclonal WF6 and polyclonal antibody, respectively. The latter was fluorescence labeled with Cy5. The different binding assays indicate the successful incorporation of the receptor in the lipid bilayer.

Kinetics of valinomycin-mediated K+ ion transport through tethered bilayer lipid membranes

Abstract Bilayer lipid membranes tethered to planar gold electrodes were prepared, based on a self assembled monolayer (SAM) of 2,3-di- O -phytanyl-sn-glycerol-1-tetraethylene glycol-DL-α-lipoic acid ester lipid (DPTL). When the SAMs were exposed to a suspension of liposomes made from diphytanoylphosphodatidyl choline (DPhyPC), tethered lipid bilayers (tBLMs) were formed with good sealing properties. The preformed tBLMs were doped with valinomycin, and the K + ion concentration in the bathing solution was increased stepwise by adding KCl. Electrical impedance spectra were recorded following every addition. These data were modelled by means of the network simulation program spice , using parameter values of the undoped membrane and a kinetic scheme for the K + /valinomycin system, whose rate constants were determined previously in independent measurements. Experimental and simulated data are in reasonable agreement despite some simplifying assumptions used in the spice simulations. Experimental data were also fitted to a conventional equivalent circuit, which reveals that the representation of the K + /valinomycin system by an ohmic resistor can be considered as no more than an approximation.

COUPLING OF PROTON TRANSLOCATION THROUGH ATPASE INCORPORATED INTO SUPPORTED LIPID BILAYERS TO AN ELECTROCHEMICAL PROCESS

Abstract H+-ATPase is incorporated into solid-supported lipid bilayers separated from the gold support by a peptide spacer. The translocation of protons across the lipid film to the inner side is coupled to the discharge of protons at the gold surface. The overall process is investigated by square wave voltammetry (SWV) and double potential-pulse chronoamperometry (CA). As a result, the formation of a proton gradient is monitored by SWV whereas currents measured by CA monitor the stationary state when the enzyme activity is directly coupled to the charge transfer at the electrode. These currents markedly depend on the number of ATPases present in the bilayer.

In Situ PM-IRRAS Studies of an Archaea Analogue Thiolipid Assembled on a Au(111) Electrode Surface

Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) has been applied to determine the conformation, orientation, and hydration of a monolayer of 2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-dl-alpha-lipoic acid ester (DPTL) self-assembled at a gold electrode surface. This Archaea analogue thiolipid has been recently employed to build tethered lipid bilayers. By synthesizing DPT(d16)L, a DPTL molecule with a deuterium substituted tetraethylene glycol spacer, it was possible to differentiate the C-H stretch vibrations of the phytanyl chains from the tetraethylene glycol spacer and acquire the characteristic IR spectra for the chains, spacer, and lipoic acid headgroup separately. Our results show that the structure of the monolayer displays remarkable stability in a broad range of electrode potentials and that the phytanyl chains remain in a liquid crystalline state. The tetraethylene glycol chains are coiled, and the IR spectrum for this region shows that it is in the disordered state. The most significant result of this study is the information that in contrast to expectations the spacer region is poorly hydrated. Our results have implications for the design of a tethered lipid membrane based on this thiolipid.

Giga-seal solvent-free bilayer lipid membranes: from single nanopores to nanopore arrays

A robust platform providing a fluid lipid bilayer is in great demand not only for specific basic research on membrane proteins, but also for related applications. Here we present electrically sealing solvent-free bilayer lipid membranes spanned over arrays of cylindrical nanopores. The nanopores are milled through thin Si3N4 diaphragms using a focused ion beam (FIB). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) reveal pores with regular shapes and inter-pore spacing. Nanopore-spanning bilayer lipid membranes (npsBLMs) are formed reproducibly by directed fusion of giant unilamellar vesicles (GUVs) to the pore-containing diaphragms. The arrays of npsBLMs exhibit electrical resistances in the GΩ range, lifetimes of up to several days, and breakdown voltages above 250 mV. Perfusion robustness of the npsBLMs and low aspect ratio of the nanopores allow easy access to both sides of the bilayers. npsBLM conductance in the presence of the pore-forming toxin gramicidin D increases depending on the concentration levels. Peptide-to-lipid molar ratios can reach as high as 1 : 23. Recordings of ionic currents through alamethicin channels are possible with single-channel resolution after dielectric passivation of the substrates. This demonstrates the applicability of the platform to biophysical research of membrane proteins as well as pharmaceutical drug screening assays.

Electronic Wiring of a Multi-Redox Site Membrane Protein in a Biomimetic Surface Architecture

Bioelectronic coupling of multi-redox-site membrane proteins was accomplished with cytochrome c oxidase (CcO) as an example. A biomimetic membrane system was used for the oriented immobilization of the CcO oxidase on a metal electrode. When the protein is immobilized with the CcO binding side directed toward the electrode and reconstituted in situ into a lipid bilayer, it is addressable by direct electron transfer to the redox centers. Electron transfer to the enzyme via the spacer, referred to as electronic wiring, shows an exceptionally high rate constant. This allows a kinetic analysis of all four consecutive electron transfer steps within the enzyme to be carried out. Electron transfer followed by rapid scan cyclic voltammetry in combination with surface-enhanced resonance Raman spectroscopy provides mechanistic and structural information about the heme centers. Probing the enzyme under turnover conditions showed mechanistic insights into proton translocation coupled to electron transfer. This bioelectronic approach opens a new field of activity to investigate complex processes in a wide variety of membrane proteins.

Fusion of small unilamellar vesicles onto laterally mixed self- assembled monolayers of thiolipopeptides

Monolayers of the thiolipopeptide NH(2)-Cys-Ala-Ser-Ala-Ala-Ser-Ser-Ala-Pro-Ser-Ser-(Myr)Lys(Myr)-OH (III) were formed on gold surfaces by self-assembly, mixed with a lateral spacer of the same peptide composition, NH(2)-Cys-Ala-Ser-Ala-Ala-Ser-Ser-Ala-Pro-Ser-Ser-Lys-OH (I). Different mixing ratios were employed ranging from 0.1 to 1, corresponding to 10-100% thiolipopeptide. These self-assembled monolayers (SAMs) were then exposed to a suspension of liposomes with the aim of forming lipid bilayers as a function of the mixing ratio. A clear optimum with respect to homogeneity and electrical properties of the membranes was obtained in the middle region (0.5) of mixing ratio, as revealed by surface plasmon resonance spectroscopy, impedance spectroscopy, and fluorescence microscopy. The combination of these methods was shown to be a powerful tool, although a true lipid bilayer was not obtained. Instead, vesicle adsorption was shown to be the predominant process, and FRAP (fluorescence recovery after photobleaching) measurements showed that the films were not fluid on the micrometer length scale.

Solid supported lipid membranes: New concepts for the biomimetic functionalization of solid surfaces

Surface-layer (S-layer( supported lipid membranes on solid substrates are interfacial architectures mimicking the supramolecular principle of cell envelopes which have been optimized for billions of years of evolution in most extreme habitats. The authors implement this biological construction principle in a variety of layered supramolecular architectures consisting of a stabilizing protein monolayer and a functional phospholipid bilayer for the design and development of new types of solid-supported biomimetic membranes with a considerably extended stability and lifetime — compared to existing platforms — as required for novel types of bioanalytical sensors. First, Langmuir monolayers of lipids at the water/air interface are used as test beds for the characterization of different types of molecules which all interact with the lipid layers in various ways and, hence, are relevant for the control of the structure, stability, and function of supported membranes. As an example, the interaction of S-layer proteins from the bulk phase with a monolayer of a phospholipid synthetically conjugated with a secondary cell wall polymer (SCWP) was studied as a function of the packing density of the lipids in the monolayer. Furthermore, SCWPs were used as a new molecular construction element. The exploitation of a specific lectin-type bond between the N-terminal part of selected S-layer proteins and a variety of glycans allowed for the buildup of supramolecular assemblies and thus functional membranes with a further increased stability. Next, S-layer proteins were self-assembled and characterized by the surface-sensitive techniques, surface plasmon resonance spectroscopy and quartz crystal microbalance with dissipation monitoring. The substrates were either planar gold or silicon dioxide sensor surfaces. The assembly of S-layer proteins from solution to solid substrates could nicely be followed in-situ and in real time. As a next step toward S-layer supported bilayer membranes, the authors characterized various architectures based on lipid molecules that were modified by a flexible spacer separating the amphiphiles from the anchor group that allows for a covalent coupling of the lipid to a solid support, e.g., using thiols for Au substrates. Impedance spectroscopy confirmed the excellent charge barrier properties of these constructs with a high electrical resistance. Structural details of various types of these tethered bimolecular lipid membranes were studied by using neutron reflectometry. Finally, first attempts are reported to develop a code based on a SPICE network analysis program which is suitable for the quantitative analysis of the transient and steady-state currents passing through these membranes upon the application of a potential gradient.