Biochimie 107 (2014) 135e142

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Mini-review

Tethered bilayer lipid membranes (tBLMs): Interest and applications for biological membrane investigations
s P. Girard-Egrot* Samuel Rebaud, Ofelia Maniti, Agne Institut de Chimie et Biochimie Mol eculaires et Supramol eculaires, Universit e Lyon 1, University of Lyon, ICBMS, CNRS UMR 5246, GEMBAS Team, ^t. Curien, 43 bd du 11 Nov. 1918, F-69622 Villeurbanne Cedex, France Ba

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 April 2014 Accepted 25 June 2014 Available online 4 July 2014

Biological membranes play a central role in the biology of the cell. They are not only the hydrophobic barrier allowing separation between two water soluble compartments but also a supra-molecular entity that has vital structural functions. Notably, they are involved in many exchange processes between the outside and inside cellular spaces. Accounting for the complexity of cell membranes, reliable models are needed to acquire current knowledge of the molecular processes occurring in membranes. To simplify the investigation of lipid/protein interactions, the use of biomimetic membranes is an approach that allows manipulation of the lipid composition of specific domains and/or the protein composition, and the evaluation of the reciprocal effects. Since the middle of the 80's, lipid bilayer membranes have been constantly developed as models of biological membranes with the ultimate goal to reincorporate membrane proteins for their functional investigation. In this review, after a brief description of the planar lipid bilayers as biomimetic membrane models, we will focus on the construction of the tethered Bilayer Lipid Membranes, the most promising model for efficient membrane protein reconstitution and investigation of molecular processes occurring in cell membranes. te  française de biochimie et biologie Mole culaire (SFBBM). All rights © 2014 Elsevier B.V. and Socie reserved.

Keywords: Lipid bilayers Biomimetic membranes Tethered bilayer lipid membrane (tBLMs) Membrane protein Biological membrane model

1. Introduction Biological membranes play a central role in cell life. Besides their function of compartmentalization, they are involved in many exchange processes between the outside and inside cellular worlds. Only a few nanometers thick, biological membranes have a perfect organization at the molecular level and consist of two main components. Lipids, held together by hydrophobic interactions, play

Abbreviations: tBLM, tethered bilayer lipid membranes; SLB, supported lipid bilayers; SAM, self-assembled monolayer; HBMs, hybrid bilayer membranes; HBMs, hydrophobic association analysis; LPS, lipopolysaccharides; npsBLMs, nanoporespanning bilayer lipid membranes; CF, protein cell-free expression; NTA, nitrilotriacetic acid; SPR, surface plasmon resonance; QCM, quartz crystal microbalance; AFM, atomic force microscopy; FRAP, fluorescence recovery after photobleaching; GPCR, transmembrane G protein-coupled receptors; PC, phosphatidylcholine; hERG, human ether-
a-go-go-related gene.  Lyon 1, Institut de Chimie et Biochimie * Corresponding author. Universite culaires et Supramole culaires (ICBMS) e UMR CNRS 5246, Equipe GEMBAS e Mole ^t Curien, 43 Bd du 11 novembre 1918, 62622 Villeurbanne, France. Tel.: þ33 4 72 Ba 44 85 32; fax: þ33 4 72 44 79 70. E-mail address: [email protected] (A.P. Girard-Egrot).

essentially a structural role by forming a self-assembled continuous bilayer acting as a diffusion barrier. Membrane proteins, like transmembrane proteins or peripheral membrane proteins, respectively embedded within the lipid bilayer or transiently associated with it, are key factors in the cell metabolism i.e. exchange or biocatalysis processes. They are involved in cellecell interaction, signal transduction, and transport of ions and nutrients. Due to these important functions, they are a target of choice for pharmaceuticals (currently more than 60% of administrated drugs). This targeting is not by chance, since nature uses the membrane as a key component allowing living cells to maintain and organize their function. Hence, unlocking the secrets of biological membranes remains a great challenge. 2. Planar lipid bilayers as biomimetic membrane models The complexity of biological membranes and their interactions with intra- and extracellular networks make their direct investigation difficult and particularly restrict in situ studies of transmembrane proteins. Therefore, the direct study of membrane proteins requires creation of rational models, which mimic the cell

http://dx.doi.org/10.1016/j.biochi.2014.06.021  te  française de biochimie et biologie Mole culaire (SFBBM). All rights reserved. 0300-9084/© 2014 Elsevier B.V. and Socie

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membrane structurally, and in which the protein retains its structural integrity and functional activity. To address this problem, a variety of biomimetic membranes have been developed with the aim to simulate the basic functions of a cell membrane and provide systems for the systematic study of different kinds of membrane-related processes. These models are solid-supported membranes [1,2], polymer-cushioned membranes [3,4], hybrid bilayer lipid membrane [5e10], free-standing lipid layer or suspended-lipid bilayers [11e13] and tethered bilayer lipid membranes [14e26]. All of these models provide the lipid environment required for the function of membrane-associated proteins. However, a suitable membrane model should fulfill other key features to mimic the natural membranes. In this context, the most appropriate route for developing membrane platforms is to design membrane structures that possess the intrinsic lateral (2-D) fluidity of the cell membrane [27]. In addition, to obtain maximum stability and to provide a suitable environment for sensing applications, the deposition of membrane models on solid supports has become very popular. The growing interest in confining lipid membranes on surfaces has been nourished by the emergence of a manifold of surface-sensitive techniques which can be applied for their own characterization [28e30] and/or the kinetic studies of biomolecular recognition [31]. In this review, we will briefly describe: i) supported-lipid bilayer; ii) polymer-cushioned membranes; iii) hybrid bilayer lipid membrane and iv) free-standing lipid layer (Fig. 1). Then, we will focus on the development of tethered bilayer lipid membranes (tBLMs) and some of their applications for biological membrane investigations. 2.1. Supported-lipid bilayers (SLBs) Among the conceptual strategies to obtain surface-confined membrane systems, the most popular is the formation of planar Supported Lipid Bilayers (SLBs). First reported by McConnell et al. [32], they are obtained by spreading small unilamellar vesicles on hydrophilic solid supports [28]. This one-step procedure, which can be achieved with different lipid mixtures [29], results in a lipid bilayer that is separated from the solid substrate by an ultrathin film of water. This layer thick (1e2 nm) confers to SLBs the fluidity required for lateral diffusion in 2D space [33]. Such a model proved well-suited to investigate lipid domain formation [34e36], membrane cell attachment [37], membrane processes such as

surface protein adsorption [38], protein self-assembly [39,40], protein localization at lipid phase boundaries [41], protein function [42] and to understand cellular organization like membraneecytoskeleton interactions [43] or immunological synapse [44e46]. However, in this system, the bilayer, in close proximity to the surface, restricts the incorporation, mobility, and consequently the stability of transmembrane proteins [47]. Indeed, the membrane-substrate distance would be too small to prevent direct contact between the solid surface and integral proteins which may possess functional units that protrude far out from the bilayer [48,49]. The limited membrane-substrate distance could lead to intense interactions and/or frictional coupling, leading to partial loss of functionality or even complete protein denaturation [50,51]. 2.2. Hybrid bilayer lipid membranes Hybrid bilayer lipid membranes have been also proposed as biomimetic membranes. Lipid monolayers are formed from small unilamellar vesicles that spontaneously adhere to a hydrophobic self-assembled monolayer (SAM), with concomitant release of their strain energy. The interaction of small phospholipid vesicles with well-characterized surfaces has been studied to assess the effect of the surface free energy of the underlying monolayer on the formation of phospholipid/alkanethiol hybrid bilayer membranes (HBMs). This approach results in the formation of a supported lipid monolayer in which the hydrophobic acyl chains of the lipids contact the hydrophobic surface, and the polar lipid head groups are presented to solution. It corresponds to the simplest method for the immobilization of half-membranes on a sensor surface by absorbing lipids onto a hydrophobic surface [5e10]. Yet, even if this approach increases the stability and robustness of the bilayer system over long time periods, a major consideration is that the lipid bilayer in HBMs remains crystalline well above the lipid bilayer melting temperature of vesicles [10]. Since the mobility of both lipids and peptides in such membranes is severely reduced, this model loses the most important property of biophysical mimics of cell membranes. Consequently, the applications of this method are limited to the investigation of receptors that are anchored in the outer leaflet of a native membrane, or to cases in which the analyte binds to the lipid itself [52]. It is the basis of HPA (Hydrophobic Association Analysis) sensor chip developed to facilitate membrane protein analysis on BIAcore™ systems. The hydrophobic association analysis or HPA chip includes a monolayer of long-chain alkanethiol groups covalently attached to the gold surface. When injected over the surface, small unilamellar vesicles containing membrane proteins rupture and fuse to form a supported lipid monolayer on the surface of the chip. This chip has been used, for instance, to analyze recombinant antibodies binding lipopolysaccharides (LPS) molecules and to demonstrate bacterial species selectivity [53]. 2.3. Polymer-cushioned membranes

Fig. 1. Models of planar lipid bilayers.

To overcome the problem of rigidity and space between the bilayer and the support, polymer-cushioned membranes have been developed, pioneered by Sackmann's group [3]. This model allows separating the membrane from the solid substrate using soft polymeric materials that rest on the substrate and support the membrane without a direct linkage. The polymer used to 'cushion' the supported membrane acts as a lubricating layer between the membrane and the substrate. This approach significantly reduces the frictional coupling between membrane-incorporated proteins and the solid support, and hence the risk of protein denaturation. This will assist self-healing of local defects in the membrane over macroscopically large substrates (cm2), and allow the incorporation

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of large transmembrane proteins without the risk of direct contact between protein subunits and the bare substrate surface [47]. During formation, when bilayers are still incomplete, defects like holes are frequently observed. The defects “healed” within about 30 min as the lipid spread. The mechanism(s) of the healing process(es) in polymer-supported bilayer system is not fully understood. One assumption made by Wagner and Tamm is that the polymers mask imperfections on the solid substrate and thereby provide a smoother and more dynamic surface for vesicle spreading [15]. Providing bilayer depleted of such defects increases the electrical resistance across the bilayer, a definite advantage for ion channel characterization. Thus, polymer-cushioned membranes allow the incorporation of large transmembrane proteins without the risk of direct contact between protein subunits and the bare substrate surface. However, in many cases, the aim of using supported lipid assemblies is to combine them with surface-based sensors to study kinetics of biorecognition reactions, but the addition of a passivation layer can sharply degrade the detection sensitivity [54]. Polymer-supported membranes have thus been widely used, mostly as a model system of the cell surface glycocalyx [3,55]. An alternative strategy, to construct cushioned membranes, involving reactive lipopolymers used as tethers [15e18] or coated on liposomes [20,21] is described later.

2.4. Free-standing lipid layer or suspended-lipid bilayers Free-standing lipid layers or suspended-lipid bilayers are membranes formed over micrometer or nanosized pores [56]. It corresponds to an attractive model with a more physiological environment for analysis since a free-standing or suspended lipid bilayer allows the membrane to be addressable from both sides of the bilayer [57]. Early efforts to create suspended lipid bilayers over micron-sized pores (so-called “black lipids” referring to the fact that they are dark in reflected light mode because they are so thin and uniform [58]) were limited by the poor stability of the suspension [59]. Recent developments in nano-fabrication have allowed the metal substrate to be machined to include nanometersized pores (~30 nm to micrometers). Lipid bilayers deposited by vesicle fusion or LangmuireBlodgett technique span these pores, which can be characterized by atomic force microscopy (AFM) indentation force as a measure of elasticity [60e62]. However, spanning all the pores of a multiporous substrate for biochip design remains a problem. Formation of nanopore-spanning Bilayer Lipid Membranes (npsBLMs) from vesicles was impaired by a high degree of randomness [63]. Danelon et al. [64] were able to spread native membranes across silicon nitride films containing apertures of 50e600 nm in diameter and total surface areas of coverage of 100 mm2. Remarkably, not only did this approach allow access to both sides of the membrane, but it preserved the native orientation of the membrane proteins. However, the problem to address protein just above the pores remains critical. In addition, the medium filling the cavities is not accessible unless the npsBLMs are disk et al. [63] formed reproducible nanoporerupted. Recently, Kresa spanning bilayer lipid membranes (npsBLMs) by directed fusion of giant unilamellar vesicles (GUVs) to the pore-containing diaphragms. The arrays of npsBLMs exhibit excellent electrical resistances in the GU range and lifetimes of up to several days. Moreover, the formation of alamethicin channel reconstituted in the suspended bilayer has permitted to record an ionic current. However, nanopore-spanning bilayer lipid membrane platforms remain for the moment, only applicable to integral channel proteins since they cannot be interfaced with detection surface sensitive-based sensors for binding recognition. Novel technology of SPR sensing scheme based on suspended lipid membranes over

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nanopores has been described, but potential applications for membrane protein investigations remain prospective [54]. 3. Concepts for tBLMs development & applications for biological membrane investigations To circumvent the problem of passivation for polymercushioned membranes or the problem of stability encountered with the free-standing bilayer, and to provide sufficient space underneath the membrane for studies of incorporated membrane proteins, various ways to anchor planar lipid membranes via chemical polymeric or oligomeric tethers have been developed. Tethered Bilayer Lipid Membranes (tBLMs) are solid supported membranes where the inner leaflet of the lipid bilayer is covalently attached to a surface through a spacer group. This spacer group covers small surface roughness features and provides aqueous reservoir with functional properties of the cytosol (or cytoskeleton). Its role is to establish a water-containing membrane space which reduces the hydrophobic influence of the metal surface, thus enabling the functional incorporation of membrane proteins. 3.1. Telechelics as spacer groups The first concept of tethering a lipid membrane to a solid support is based on the use of anchor lipids also called telechelics [14]. They are composed of three distinct molecular parts: i) an amphiphile that becomes part of the proximal monolayer of the final bilayer structure (anchor lipid), ii) a spacer unit, the “tether” that decouples the bilayer from the substrate, iii) a substrate-specific head group, for instance based on thiol, disulfides, lipoic acid,

Fig. 2. The assembly process leading to the formation of a tethered bilayer lipid membrane from telechelics as spacer groups. Telechelic amphiphiles self-assemble to form a tethered monolayer onto a clean Au substrate (used for surface plasmon- and impedance spectroscopic characterization of the assembly process, for instance). The bilayer is then completed by vesicle fusion on the proximal monolayer. Protein incorporation into the tethered membrane could be achieved as further expectation by fusion of proteo-liposomes or ensured upon detergent removal (adapted from Knoll et al. [65]).

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silane, or alike for gold, silver, silicon oxide, metal supports, or mercury electrode [22,25,51,65] (Fig. 2). Anchor lipids are mainly archaea analog thiolipids with two phytanoyl chains that improve the fluidic (hence, sealing) character of the hydrophobic core and polyethylene glycol as spacer [24,66e70], telechelic PEG lipopolymer [15e19,71] or lipoglycopolymer [72]. Evans's group has proposed to use cholesterol-based anchors [73]. Recently, Basit et al. [27] have synthesized a novel anchoring thiol, namely 2,3-dio-palmitoylglycerol-1-tetraethylene glycol mercaptopropionic acid ester (TEG-DP); Fabre et al. [74] have used skeletonized zirconium octadecylphosphonate surface as support. The process of the functional membrane fabrication starts with the assembly of telechelic lipid derivatives that are designed to covalently bind to and self-organize at the solid support from solution, thus forming the proximal monolayer of the final architecture. After fusion of vesicles from a liposomal dispersion in contact to this monolayer, the tethered Bilayer Lipid Membranes (tBLMs) was formed. This can be then characterized by a broad range of surface analytical techniques, like Surface Plasmon Resonance (SPR) [24,65] and IR spectroscopy [75], and Quartz Crystal Microbalance (QCM) [24]. This approach is further expected to allow protein incorporation by fusion of vesicles that could contain reconstituted proteins (Fig. 2). However, limited integration capacity for membrane proteins, particularly those composed of large subunits has been demonstrated in the case of tBLMs from telechelics [51]. Generally speaking, tBLMs can partially overcome the challenge of losing protein diffusion or inducing protein denaturation owing to friction to the support by lifting the membrane a few nanometers above the substrate. Spacers, like anchors lipid, could require difficult chemistry [54]. 3.2. Peptide-tethered bilayer lipid membranes (pep-tBLMs) A modified strategy uses the assembly of peptides as spacers to form peptide-tethered lipid bilayer membrane (pep-tBLMs) (Fig. 31). The peptides are typically attached to a gold substrate by sulfhydryl or lipoic acid groups at the N-terminus while their C-terminus is being activated afterward. Peptide self-assembled monolayers used as tether are prepared from synthetic or native thiopeptides, or thiolipopeptides [51]. Different peptides, functionalized with terminal sulfur groups such as cysteine or lipoic acid designed for self-assembly on gold, have been used to prepare pep-tBLMs (Table 1), the building-up being mainly followed by SPR spectroscopy [76], Fluorescence Recovery After Photobleaching (FRAP) and characterized by Atomic Force Microscopy (AFM) [77]. The terminal carboxyl group of thiopeptides P5, P7, P19 is used to covalently attach the amino group of dimyristoylphosphatidylethanolamine (DMPE), using NHS/EDCactivated carboxyl group to form an ester linking, thus forming a peptide-tethered lipid monolayer. P19 is a water-soluble peptide derived from a-laminin subunit (laminin is a complex glycoprotein of extracellular matrix, consisting of three chains a, b and g able to interact with integrins and implies in the cell attachment, differentiation, cell shape and movement). Pep-tBLM formed with this natural thiopeptide offers the best hydrophilic submembrane part. The thiolipopeptide LP12 is used like this. Subsequently, a lipid bilayer is formed by fusion of liposomes with or without the reconstituted protein of interest. By this way, cytochrome c oxydase, Hþ-ATPases (from chloroplasts and Escherichia. coli) or the dimer of nicotinic acetylcholine receptor have been successfully reincorporated in pep-tBLMs formed with P5, P7 or the thiolipopeptide LP12, and interaction study of antagonists has performed by surface plasmon spectroscopy [78e82]. Sinner et al. have been functionally incorporated

Fig. 3. Examples of different ways for forming tethered lipid bilayer membranes (tBLMs). (1) Self-assembly of peptide used as spacer covalently coupled with anchor lipid followed by vesicle spreading to form a complete bilayer (2) tBLMs obtained from liposomes containing DSPE-PEG-NHS anchorage to an amine-coated surface. The bilayer is then completed by vesicle fusion (3) avidin-tethered lipid bilayer made from the attachment of biotinylated vesicles on an avidin layer previously formed onto a layer of biotinylated BSA and then disrupted to form the bilayer.

integrins (cell-adhesion receptor) into P19-tethered bilayer lipid membrane, and successfully investigated integrin-ligand interactions [83]. P19-tethered bilayer lipid membranes have been also used as a model system for in situ cell-free (CF) expression proteins. The idea is to bypass the difficult expression, purification and reconstitution procedures inherent when dealing with complex membrane proteins. By combining cell-free expression of proteins and pep-tBLMs, Robelek et al. [84] have obtained a spontaneous and vectorial insertion of odorant receptor OR5, a member of the seven-transmembrane G protein-coupled receptors (GPCR) family, into peptide-tethered membrane composed of P19/DMPE and soybean phosphatidylcholine (Immunolabeling with antibodies against amino and carboxy terminal tags validated that amino terminus of OR5 was outside the membrane). The in vitro insertion of the protein in the membrane is achieved by a coeor posttranslational process into the peptide-tethered membrane mimicking the biological membrane. Recently, Sinner et al. applied the same strategy to reincorporate a protein complex, namely cytochrome bo3 ubiquinol oxidase [85,86] or hERG (human ether
a-go-go-related gene) potassium channel [87]. In both cases, they

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Table 1 Amino acid sequences and thio- and thiolipeptides used for coupling membranes to solid substrates (from Sinner et al. [51]). Thiopeptide Thiopeptide Thiopeptide Thiopeptide

P5: P7: P19: LP12:

Lip-Ala-Ala-Ala-Ala-Ala-COOH HS-(CH2)2-Ala-Ser-Ser-Ala-Ala-Ser-Ala-COOH HS-Cys-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-Asp-Arg-COOH HS-Cys-Ala-Ser-Ala-Ala-Ser-Ser-Ala-Pro-Ser-Ser-Lys(Myr)-Myr

Abbreviations: ‘Lip’ for lipoic acid; ‘Myr’ for myritoic acid, (C14).

have demonstrated the functionality of the incorporated proteins, and in the latter case, they are capable in detecting interactions between hERG and different channel blockers. Hence, pep-tBLMs are shown to be well-suited for membrane protein incorporation in an active form with the advantage of providing biocompatible spacer moiety. They are well designed to preserve the integrity and functionality of membrane proteins. Finally, Song et al. [88] have proposed to use combined LangmuireBlodgett and Langmuir-Schaefer transfers to form peptidetethered membranes. In this approach, a monolayer of DMPE was directly transferred by vertical dipping onto activated carboxyl groups of a P19 self-assembled monolayer formed on gold by strong SeAu interaction. The distal lipid monolayer was obtained by Langmuir-Schaefer transfer. Owing to this stepwise assembly, it is easy to change the lipid composition of the distal layer. By this way, they have analyzed the adsorption of the amyloid b-peptide (Ab40) with several lipid mixtures. They found that the Ab adsorption is critically depending on the lipid composition of the membranes, with Ab specifically binding to membranes containing sphingomyelin. Further, this preferential adsorption was markedly amplified by the addition of sterols. In parallel, Becucci et al. [89] have incorporated channelforming peptides, gramicidin and alamethicin, in a mercurysupported lipid bilayer composed of a tethered thiolipid monolayer with a self-assembled dioleoylphosphatidylcholine (DOPC) monolayer on top of it. The thiolipid consists of a hexapeptide chain with a high tendency to form a 310-helical structure, which terminates at the N-terminus end with a sulfhydryl group for anchoring to the metal, while the C-terminus end is covalently linked to the polar head of DMPE. The hexapeptide moiety has two triethyleneoxy side chains that impart a satisfactory hydrophilicity and are intended to keep the anchored thiolpeptide chains sufficiently apart, so as to accommodate water molecules and inorganic ions and to create a suitable environment for the incorporation of integral proteins. All together, these promising results show that peptide-tethered membranes provide a biomimetic model which is appropriate for the functional insertion of membrane proteins of different species and complexity. However, whatever the method used, the type and the length of the spacer that gives physico-chemical properties to the submembrane part, and the nature of the anchor itself has a significant influence on the properties of tBLM [69]. The ability to flexibly adjust spacer length and lateral spacer density makes it possible to finely tune the membraneesubstrate distance and the viscosity of the bilayer, both of which control the lateral diffusivity and function of transmembrane proteins [47].

is completed by deposition of vesicles containing a mixture of PC and DSPE-PEG-NHS, which acts both as a spacer and an anchor molecule. The spacer lipid is terminated by an NHS activated carboxylic acid that can react with any amine-coated surface (cysteamine-coated gold or amino-silanized glass). Egg PC or POPC/DSPEPEG-NHS mixture liposomes are injected on the top of an amine grafted surface. After linking of vesicles to the surface, their disruption leads to the formation of a bilayer. Formation of the supported bilayers on gold surfaces has been followed using SPR spectroscopy [20] and characterized by AFM [26]. Studies of the diffusion coefficient of individual lipids within the bilayer by fluorescence recovery after photo-bleaching (FRAP) measurements have indicated that the bilayer is fluid and continuous to preserve the high lateral mobility of membrane constituents despite the proximity of the solid support [20,26]. An important advantage of the direct incorporation of the spacer molecule in vesicles is the possibility to control its percentage. This allows obtaining a spacer layer with low density in comparison to very closely packed layers of models which are supported by a self-assembled supported anchor lipid monolayer [24,66,71,90]. Indeed, high spacer densities could constitute a steric limitation and proteins having large extramembrane domains cannot be incorporated appropriately. This type of bilayer has been successfully used as a biomimetic membrane for protein/membrane interaction investigations of different kinds of integral proteins : the bacterial toxin, the adenylate cyclase produced by Bordetella pertussis (CyaA) [20], the mitochondrial outer membrane channel, the voltage dependent anion channel (VDAC) [26] and the Aquaporin Z (AqpZ); this latter being incorporated in the tBLM by cell-free expression system [91]. Recently, Rossi et al. have improved the model of tBLMs formed by polymer-coated liposomes spreading [92]. Using this approach, they have designed a tethered lipid bilayer assembled on the top of an amine-gold surface derivatized with calmodulin serving as specific cytoplasmic marker, as a proof of concept that tBLM may be useful to mimic cellular compartmentalization. With this sophisticated model, they have demonstrated the calcium-dependent translocation of catalytic domain of the toxin CyaA (adenylate cyclase from B. pertussis) across the tBLM by applying a negative membrane potential [93]. In this model, the translocation has been evidenced by the binding of CyaA domain to the immobilized calmodulin, revealed by enzymatic assay. Hence, it appears that such a biomimetic tBLM construction should provide opportunities to explore the molecular mechanisms of protein translocation across biological membranes in precisely defined in vitro conditions. 3.4. Avidin/biotin system as spacers for tBLMs

3.3. tBLMs formed by polymer-coated liposomes spreading on planar substrates Besides anchor lipids and peptide spacers, another concept for forming tethered membranes is based on the direct fusion of vesicles that contain both the anchor and the spacer molecules on a functionalized surface [20,21] (Fig. 3-2). In brief, surface coating is obtained from self-assembled monolayers containing amino groups (amino-thiols or aminosilanes). The bilayer formation step

A similar approach has used affine avidin/biotin system as spacer for tethering membranes (Fig. 3-3). In a first step, intact biotinylated vesicles are accumulated by affinity on a sublayer of immobilized streptavidin. After rinsing, the formation of the bilayer is triggered by addition of soluble poly(ethylene glycol) (PEG), a fusiogenic agent of lipid vesicles [94,95]. A crucial step in the formation of tBLMs is the achievement of spontaneous vesicle fusion on the template, in such a way that a continuous fluid bilayer

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without defects can be produced in a reasonable time, compatible with the survival of the native membrane proteins. This two-step strategy for the assembly of a supported bilayer on a streptavidin sublayer overcomes the constraints imposed by an uncontrolled kinetics of spontaneous vesicle fusion [96,97]. By this approach, Elie-Caille et al. [98] have successfully tethered and fused phospholipid-enriched proteoliposomes prepared from the inner mitochondrial membrane and containing the transmembrane proteins of the respiratory chain at a protein area fraction of about 15%. Step-by-step assembly of the structure and triggered fusion of the immobilized (proteo-)liposomes have been monitored by SPR and FRAP, respectively. A homogenous flat surface of soft material corresponding to a biomimetic (proteo-)lipid bilayer anchored to the solid substrate has been formed [96,98]. Recently, Sumino et al. [99] used the avidinebiotin interaction to tether lipid bilayer containing photosynthetic antenna proteins (LH2- and LH1-RC) for functional analysis. Energy transfer from LH2 to LH1-RC within the tethered membrane, observed by steady-state fluorescence spectroscopy and due to the diffusivity of the proteins inside the bilayer, indicates that biotinylated planar membranes tethered by pillars of avidin molecules immobilized onto the substrate can mimic the natural situation. 3.5. Other strategies Another strategy has been proposed to develop tethered biomimetic membranes. It concerns the attachment of membrane proteins solubilized in detergent micelles on the support before lipid bilayer formation around bound proteins by in situ dialysis in the presence of liposomes [100,101]. For this purpose, His-tagged proteins are directly coupled to the support by the way of chelating nitrilotriacetic acid (NTA) groups grafted on the solid surface in the presence of Cu2þ or Ni2þ. This approach leads to the concept of protein-tethered lipid bilayer membranes (ptBLM) first developed by Geiss et al. [102]. The generation of the ptBLM is completed by replacing detergent molecules of the bound protein by lipid, thus forming small patches of lipid bilayers between the protein molecules, which eventually would seal the whole assembly to a closed membrane. This strategy allows controlling the orientation and the packing density of the reconstituted proteins in the lipid bilayer. In contrast, it is only relevant for studies of membrane properties or protein functions in which the lateral mobility of the protein is irrelevant since all the proteins are immobilized on the surface [51]. Given examples are redox proteins, like cytochrome c oxydase, that one might want to connect electronically to the base electrode, because they need to be ‘wired’ to the support for an efficient heterogeneous electron transfer between the external circuit and the redox center of the protein [103e105]. Recently, Boxer et al., [106,107] developed a method for spatially separating a lipid bilayer from a solid support using DNA-modified lipids. Surface-reactive DNA sequences are spotted onto a glass slide, and giant unilamellar vesicles containing covalently attached antisense DNA strand are added to form patches on the complementary printed DNA. However, the surface passivation is currently insufficient to completely prevent GUVs from leaving small amounts of contamination on the surface. Nevertheless, with further development, this approach would enable rapid screening of different patches in protein binding assays and may enable the incorporation of membrane proteins. 4. Conclusion and prospective remarks The achievement of tBLMs is a thriving way to mimic the membrane environment for transmembrane protein reconstitution and functional investigation. Several studies using tBLMs have

allowed the reconstitution of complex mechanisms in membrane models, involving several cell signaling molecules. These studies mainly focus on the formation of supramolecular assemblies based on the lateral diffusion of extracellular domains of membrane receptors and other protein-related interactions. Still very few models have been developed to take into account the phenomena occurring on the intracellular side. This aspect seems important, especially in the study of signal transduction to characterize the events occurring at the membrane itself, but also to track responses or mechanisms directly generated at the intracellular level. Creating tBLMs remains yet complex and almost a case-by-case development. Currently, there is no method for the preparation of versatile tBLMs allowing the construction of a tethered polyvalent lipid bilayer constituted of heterogeneous lipid compositions in order to better mimic the natural composition of biological membranes. Up to now, the method employed to form tBLMs limits the phospholipid composition of the bilayer due to technical constraints which impose the use of certain lipids (see above). Filling this gap and becoming able to develop tBLM of any phospholipid composition may be the key for a generic insertion of any membrane proteins into tethered membrane platforms. The wide range of biotechnological applications relying on biomimetic membranes confined to planar surface-sensitive supports gives rise to the huge promise that these technologies hold for the next decade. Transmembrane proteins are targeted by nearly 60% of all drug candidates. Therefore, membrane chip design containing transmembrane proteins for high throughput studies represents a promising method for the screening of pharmacologically membrane active compounds with potential benefits in the development of new drug candidates or diagnostic test systems. Nowadays, the realization of membrane chips as drug screening platforms depends critically on i) the capacity to efficiently produce and immobilize membrane proteins of interest, especially due to the difficulty to handle that kind of proteins and to reconstitute them into hydrophobic lipid environment, and ii) the stability of the supporting membrane [108]. Indeed, the study and the exploitation of membrane protein for drug screening applications require a controllable and reliable method for their delivery into an artificial membrane platform based on lab-on-chip technology. To date, tBLM remains the most attractive approach to develop such a biomimetic system, which improve the stability of the membrane proteins and their applicability for sensing assay, and thus remove the technological barrier. Conflict of interest There is no conflict of interest. References [1] A.A. Brian, H.M. McConnell, Allogeneic stimulation of cytotoxic T cells by supported planar membranes, Proc. Natl. Acad. Sci. 81 (19) (1984) 6159e6163. [2] L.K. Tamm, H.M. McConnell, Supported phospholipid bilayers, Biophys. J. 47 (1) (1985) 105e113. [3] E. Sackmann, Supported membranes: scientific and practical applications, Science 271 (5245) (1996) 43e48. [4] E. Sackmann, M. Tanaka, Supported membranes on soft polymer cushions: fabrication, characterization and applications, Trends Biotechnol. 18 (2) (2000) 58e64. [5] A.L. Plant, Self-assembled phospholipid/alkanethiol biomimetic bilayers on gold, Langmuir 9 (11) (1993) 2764e2767. [6] J.K. Cullison, et al., A study of cytochrome c oxidase in lipid bilayer membranes on electrode surfaces, Langmuir 10 (3) (1994) 877e882. [7] J.D. Burgess, M.C. Rhoten, F.M. Hawkridge, Cytochrome c oxidase immobilized in stable supported lipid bilayer membranes, Langmuir 14 (9) (1998) 2467e2475. [8] V.I. Silin, et al., The role of surface free energy on the formation of hybrid bilayer membranes, J. Am. Chem. Soc. 124 (49) (2002) 14676e14683.

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