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New tethered phospholipid bilayers integrating functional G-Protein Coupled Receptor membrane protein Meriem Chadli, Samuel Rebaud, Ofelia Maniti, Bruno Tillier, Sandra Cortès, and Agnes Girard-Egrot Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01636 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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New tethered phospholipid bilayers integrating functional G-Protein Coupled Receptor membrane protein Meriem Chadli1,2†, Samuel Rebaud1†, Ofelia Maniti1, Bruno Tillier2, Sandra Cortes2, Agnès GirardEgrot1* 1

Univ Lyon, Université Lyon 1, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, ICBMS, UMR CNRS 5246, 43 Bd du 11 Novembre 1918, F-69622 VILLEURBANNE, France 2 Synthelis, Biopolis, 5, avenue du Grand Sablon, 38700 LA TRONCHE, France †

These authors have equally contributed to this study

ABSTRACT

Membrane proteins exhibiting extra- and intra-cellular domains require an adequate near-native lipid platform for their functional reconstitution. With this aim, we developed a new technology enabling the formation of a peptide-tethered Bilayer Lipid Membrane (pep-tBLM), a lipid bilayer grafted onto peptide spacers, by the way of a metal-chelate interaction. To this end, we designed an original peptide spacer derived from the natural α-laminin thiopeptide (P19), possessing a cysteine residue in N-terminal extremity for grafting on gold and modified in C-terminal extremity by four histidine residues (P19-4H). In the presence of nickel, the use of this anchor allowed to bind liposomes of variable compositions containing 2% molar ratio of a chelating lipid, 1,2-dioleoyl-snglycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] so called DOGS-NTA, and to form the planar bilayer by triggering liposome fusion by an α-helical (AH) peptide derived from the N-terminus of the hepatitis C virus NS5A. The formation of pep-tBLMs was characterized by Surface Plasmon Resonance imaging (SPRi), and their continuity, fluidity and homogeneity were demonstrated by Fluorescence Recovering After Photobleaching (FRAP), with a diffusion coefficient of 2.5.10-7 cm²/s, and Atomic Force Microscopy (AFM). By using variable lipid compositions including Phosphatidylcholine

(PC),

Phosphatidylserine

(PS),

Phosphatidylethanolamine

(PE),

Phosphatidylinositol 4,5-bisphosphate (PIP2), Sphingomyelin (SM), Phosphatidic Acid (PA) and Cholesterol (Chol) in various ratios, we show that the membrane can be formed independently from the lipid composition. We made the most of this advantage to reincorporate a transmembrane protein in an adapted complex lipid composition to ensure its functional reinsertion. For this purpose, a cell-free expression system was used to produce proteoliposomes expressing functional C-X-C motif chemokine receptor 4 (CXCR4), a seven-transmembrane protein belonging to the large superfamily of G-protein-coupled receptors (GPCRs). We succeeded in reinserting CXCR4 in peptBLMs formed on P19-4H by fusion of tethered proteoliposomes. AFM and FRAP characterizations allowed us to show that pep-tBLMs inserting CXCR4 remained fluid, homogeneous and continuous. 1 ACS Paragon Plus Environment

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The values of the diffusion coefficient determined in the presence of reinserted CXCR4 was 2.10-7 cm²/s. Ligand binding assays using a synthetic CXCR4 antagonist, T22 ([Tyr5,12, Lys7]-polyphemusin II) revealed that CXCR4 can be reinserted in pep-tBLMs with a functional folding and orientation. This new approach represents a method of choice for investigating membrane protein reincorporation and a promising way of creating a new generation of membrane biochips adapted for screening agonists or antagonists of transmembrane proteins.

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I.

INTRODUCTION

Membrane proteins play a major role in every living cell. They are involved in many extra and intracellular processes, like cell adhesion, ion transport, signal transduction and cellular metabolism. Hence, understanding protein-protein and ligand-receptor interactions within the cellular membranes remains a fundamental aspect of cellular biology. Additionally, due to their essential functions, transmembrane proteins are currently targeted by more than 60% of commercialized drugs1 and hence, are of great interest for pharmaceutical research. Screening new therapeutic molecules involves studying membrane proteins functions as well as detecting and analyzing their interactions with agonist or antagonist ligands. However, production, purification and functional reconstitution of membrane proteins remain challenging. Due to their amphiphilic nature, membrane proteins are difficult to extract and are subject to early denaturation upon their manipulation. Their fragile nature requires a near native lipid environment to properly evaluate their functionality. In this context, biomimetic lipid membranes formed on solid supports allowing functional membrane protein insertion have emerged as a key model for both fundamental study of biological membranes and drug screening targeting membrane proteins. Different types of models have been developed to mimic cell membranes, including solidsupported membranes,2-3 polymer-cushioned membranes,4-6 hybrid lipid bilayers,7-9 free-standing lipid layer or suspended-lipid bilayers,10-12 and tethered bilayer lipid membranes (tBLMs).13-24 Among them, tBLMs have emerged as very attractive platforms to provide stable and fluid bilayer membranes adapted for membrane protein insertion. tBLMs are in fact a natural progression from the planar supported lipid bilayers (SLBs). First reported by McConnell et al.,3 SLBs are classically obtained by the spreading of small unilamellar vesicles on hydrophilic solid supports25-27 and result in a lipid bilayer separated from the solid substrate by an ultrathin film of water (1-2 nm), which confers them the fluidity required for lateral diffusion in 2D space.25 However, in this system, the close proximity of SLBs to the substrate restricts the incorporation of transmembrane proteins, which may possess functional units that protrude far out from the bilayer.28 Membrane proteins incorporated in SLBs suffer from nonphysiological interactions with the solid support,4 leading both to a loss of protein dynamics and a partial loss of functionality, or even complete protein denaturation.29-31 This problem is particularly serious when working with membrane proteins, whose functional extra- and intra-cellular domains can extend to several tens of nanometers. Consequently, it is crucial to decouple the lipid bilayer from the supporting solid substrate to minimize interactions of the protein with the substrate, and to provide sufficient space for the correct conformation of transmembrane domains. In this context, tBLMs spaced from the surface by a tethering molecule, e.g. polymer cushion,23,28,32-34 telechelics,22,35 peptides29,36 or proteins,37-44 circumvent this problem by lifting the 3 ACS Paragon Plus Environment

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membrane off the surface and providing an additional space, thus enabling functional incorporation of transmembrane proteins and satisfying to requirements of ruggedness, fluidity, and eventually, high electrical resistance, which are necessary for the incorporation of integral membrane proteins in a functionally active state.45 A tBLM consists of a lipid bilayer, where the proximal leaflet is covalently bound to a solid support via a soft, flexible and hydrophilic spacer group.15,18,21,28-30,45-46 The role of the spacer group is multiple. It covers small surface roughness features, provides a hydrated reservoir between the substrate and the membrane, reduces the hydrophobic influence of the metal surface and unfavorable frictions to the support, supplies ample space to accommodate protein ectodomains, thus keeping the mobility and the flexibility of the reinserted membrane proteins. In addition of maintaining fluidity, the chemical robust attachment of the tethered membranes to the surface offers a very stable system, which makes it possible to perform experiments over extended time periods and gives access to a manifold of surface analytical techniques, such as atomic force microscopy (AFM) or surface plasmon resonance imaging (SPRi).42,47-48 Various strategies prevail in the literature to achieve tBLMs. Among them, polar peptides used as tethers to form peptide-tethered lipid bilayers (pep-tBLMs) are shown to be suited for membrane protein incorporation by providing a biocompatible spacer moiety in which the membrane proteins can fold in a native-like functional conformation. The main advantage of using peptides as tethers is that their length, their secondary structure and their hydrophilic properties can be easily tuned by changing their aminoacid sequences. This enables a flexible adjustment of both membrane–substrate separation and viscosity of the tethering units, which are important to ensure sufficient lateral membrane diffusivity for functional protein incorporation.28 Classically, peptides used as tethers to form pep-tBLMs are prepared from synthetic or native thiopeptides, or thiolipopeptides.29 These peptides are functionalized in N-terminus by a sulfur group such as a cysteine, or a lipoic acid designed for self-assembly on gold, a surface of choice for biosensing applications, while their C-terminus extremity is being chemically activated afterward for the coupling of the amino polar headgroup of different phosphatidylethanolamine (PE); thus forming the proximal lipid monolayer,21 which acts as the anchor of the membrane by forming the proximal leaflet of the bilayer.36,49-52 This first layer is built via self-assembly process from a dilute solution of tether molecules. The bond with the substrate provides stable anchoring of the proximal leaflet. Subsequently, the distal layer is generally formed by fusion of liposomes with or without the reconstituted protein of interest onto the hydrophobic self-assembled proximal layer.30 By this way, different integral membrane proteins e.g. cytochrome c oxidase,36 cytochrome bo3 ubiquinol oxidase,53 H+-ATPases (from chloroplasts and E. coli),49-50,52 dimer of nicotinic acetylcholine receptor from Torpedo californica,51 hERG potassium channel54 or channel-forming peptides like gramicidin or 4 ACS Paragon Plus Environment

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alamethicin55 have been successfully reincorporated in pep-tBLMs formed with functionalized thio(lipo)peptides. In the classical way to prepare pep-tBLMs, the bilayer is formed in two steps, both leaflets being formed independently: first proximal layer by self-assembly process, and second distal layer completed by fusion of vesicles. Nevertheless, it has been reported that this two-step procedure led sometimes to a heterogeneous and/or discontinuous tethered lipid bilayer due to the partial surface coverage with some non-disrupted liposomes adsorbed onto the solid surface.21,29,56 Additionally, this leaflet-by-leaflet formation process appears less suitable regarding the reincorporation of transmembrane proteins that require a lipid core for maintaining their function, these latter being able to be denatured during the coverage of the hydrophobic proximal layer by the distal monolayer. In order to address this problem, some authors have proposed to incorporate integral membrane proteins by cell-free expression synthesis directly onto preformed tBLMs, whatever the tethering molecules used e.g. polyethylene glycol (PEG),23 telechelics,22 peptides53-54,57 or proteins.58 By this way, a number of membrane proteins have been successfully reincorporated in pep-tBLMs,53-54 and for some of them, the orientation has been assessed by immunolabeling and/or surface plasmon enhanced fluorescence spectroscopy (SPFS).53,57 However, from a biochemical point of view, a proper reinsertion or reincorporation of transmembrane proteins in a lipid bilayer is not a guarantee of the functional folding of the protein, and only few report the functionality of the reinserted membrane protein in tBLMs.22,57 A possible explanation of this failure is the small amount of reincorporated proteins,53 which does not allow to measure any protein activity. Additionally, the functionalization of the thiopeptides by the lipid anchor to generate lipopeptides used in the classical way to prepare pep-tBLMs requires a synthetic chemistry, often complicated, which limits the type of lipid anchor which can be added, classically dimyristoylphosphatidylethanolamine (DMPE).21 Hence, the proximal layer is monospecific and only formed by one lipid species, which could restrict both the dynamic behavior of the tethered lipid bilayer and the capacity of the membrane protein to be reinserted in this latter; the composition of the bilayer in terms of length and/or unsaturation of the fatty acyl chains on the one hand, and the nature of the phospholipid polar group on the other hand, being capital for a guarantee of a successful functional reincorporation. In this work, we propose an alternative approach to prepare pep-tBLMs and form the bilayer in a single step, thus avoiding any chemistry. With this approach, liposomes including or not reconstituted membrane proteins are attached to the grafted peptide spacers by a metal-chelate interaction before triggering their fusion by a fusogenic agent to form pep-tBLMs (Figure 1 A & B). The metal-chelate interaction has already been used to graft membrane proteins solubilized in detergents to chemically modified gold surface via the affinity of its histidine-tag for a nickelchelating nitrilo-triacetic acid (NTA) surface before reconstituting them into the lipid environment by 5 ACS Paragon Plus Environment

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detergent substitution,59 but never for attaching the lipid membrane onto grafted peptide spacers. Here, we have designed a new peptide spacer derived from the natural thiopeptide (P19) and modified in its C-terminal extremity by four histidine residues (P19-4H, Figure 1C) to bind liposomes containing 2% of a chelating lipid (DOGS-NTA, Figure 1D).60 P19 is a water-soluble peptide derived from the subunit α of laminin, an extracellular matrix glycoprotein implied in the cell-cell interaction and cell differentiation. It is one of the most widely used peptides to prepare pep-tBLMs since its Nterminal cysteine residue allows spontaneous grafting on gold surface via stable Au-thiolate bond formation, and its natural composition offers an adequat hydrophilic sub-membrane environment. It has been previously used for incorporating integrins (cell-adhesion receptors) and successfully investigating integrin-ligand interactions,61 or for inserting cell-free expressed G protein–coupled receptors (GPCRs), and others.21,53-54,57 DOGS-NTA presents a chelating headgroup that forms a coordination complex with histidine residues in the presence of nickel with a high binding affinity (Kd = 10−13 M for a six residue polyhistidine tag at pH 8.0).62 Suitable to coat materials, chelating lipids have been used to immobilize proteins and functionalize different systems, like lipid surfaces for bioreceptor immobilization.63-64 The main interest of using chelating lipids for surface functionalization lies on the versatility and the reversibility of the immobilization in the presence of imidazole. Here, the mode of attachment by chelation between P19-4H and the headgroup of DOGSNTA will confer a great stability during handling compared to other pep-tBLMs described in the literature, in which the lipid bilayer is only anchored by hydrophobic interaction (i.e. hydrophobic chains of DMPE attached to the C-terminal end of peptide spacer inserted inside the bilayer). Indeed, it is well-known that physisorbed membranes to the support can eventually lead to delamination and partial destruction of the membrane architecture. Tethered liposomes are then fused by an amphipathic agent to obtain a planar membrane, as previously proposed by Berquand et al. in another system, e.g. to fuse liposomes attached to aluminum oxide by the way of avidin/streptavidin system.41 In the present study, we have used an amphipathic α-helix (AH) peptide derived from the N-terminus of the hepatitis C virus NS5A protein65 to induce vesicle rupture and subsequent peptBLM formation. This peculiar peptide has been previously shown to induce bilayer formation on gold substrate,66-69 and more recently, to form tBLMs on polymer (PEG) cushion23 or mesoporous silica.70 The AH peptide binds to the vesicle surface, promotes vesicle swelling, and then desorbs, leading to the formation of a lipid bilayer.71-72 The main original feature of the methodology proposed here, compared to the classical way to form pep-tBLM, is that we use preformed entire vesicles before fusion, and then, the lipid composition of the bilayer can be easily tuned and complexified. Hence, it can be adapted for (trans)membrane protein reinsertion since it will offer the possibility to reincorporate them in a native-like lipid environment, which is essential to keep their natural conformation and their biological activity on a gold surface for performing ligand binding assays. 6 ACS Paragon Plus Environment

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In the present work, we have first characterized the formation of pep-tBLMs of variable lipid compositions by surface plasmon resonance imaging (SPRi), and their homogeneity, lateral uniformity and fluidity have been checked by fluorescence recovering after photobleaching (FRAP) and atomic force microscopy (AFM). Then, we have transposed this approach to the fusion of proteoliposomes inserting a functional C-X-C motif chemokine receptor 4 (CXCR4), a seventransmembrane protein belonging to the large superfamily of G-protein-coupled receptors (GPCRs). Expression and biophysical studies of GPCRs are very challenging. Indeed, the production of recombinant membrane proteins by classical expression systems presents some limiting features on several aspects: obtaining of reasonable quantity of functional membrane proteins and it is even impossible to produce cytotoxic proteins. A very interesting and attractive alternative is the use of cell-free transcription/translation systems. Synthelis SAS has developed and optimized a new membrane protein cell-free expression system to provide several milligrams of functional GPCRs such as CXCR4. This method is based on the in vitro expression of the membrane proteins using a cell-free system in presence of liposomes which are added to the reaction medium. In a single stage reaction, CXCR4 are integrated into a defined lipid bilayer to produce active CXCR4 proteoliposomes directly.7376

This chemokine CXCR4 was chosen as a model because of its relevance in pharmaceutical research.

Many chemokine signaling pathways are also vital for cell migration in normal development or in abnormal conditions such as tumor metastasis. For instance, the CXC chemokine stromal cell derived factor-1 (SDF-1, also known as CXCL12) and its receptor CXCR4 are essential for proper fetal development. CXCR4 is also the major coreceptor for T-tropic (X4) strains of human immunodeficiency virus 1 (HIV-1), and SDF-1 inhibits HIV-1 infection. Additionally, SDF-1 and CXCR4 mediate cancer cell migration and metastasis. CXCR4 is found in cells from over 20 types of cancers, which metastasize to tissues that secrete SDF-1, including the bone marrow, lung, liver, and lymph nodes.77-79 Hence, CXCR4 represents an important therapeutic target and its functional reinsertion in pep-tBLMs for investigating ligand binding will bring a better understanding of the modulation of chemokine-receptor function by different antagonists. Our results show that CXCR4 can be efficiently reinserted in a pep-tBLMs of a complex lipid composition. The ligand binding assays performed before and after fusion of tethered proteoliposomes integrating CXCR4 have confirmed that the new methodology developed in this study allows the integration of a transmembrane protein within the reconstituted membrane with a functional folding and orientation. To our knowledge, CXCR4 has never been functionally integrated in a planar lipid bilayer before. Thus, we believe that our current approach could be applied to other membrane proteins for drug screening or other protein-related interactions.


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II.

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EXPERIMENTAL SECTION

1. Reagents. Phosphate 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer and NaCl were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). DOPC (1-palmitoyl-2-oleoyl-snglycero-3-phosphocholine, MW: 760.076), DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt), MW: 810.025), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, MW: 744.034), DMPA (1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt), MW: 614.767), Chol (cholesterol, ovine, average MW: 386.654), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, MW: 760.076), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, MW: 717.996), POPS (1-palmitoyl-2oleoyl-sn-glycero-3-phospho-L-serine (sodium salt), MW: 783.988), PI(4,5)P2 (1,2-dioleyl-sn-glycero3-phosphoinositol-4,5-bisphosphate, MW: 1074.158), egg PC (L-α-phosphatidylcholine (Egg, Chicken, average MW: 770.123), Brain PS (L-α-phosphatidylserine (Brain, Porcine) (sodium salt), average MW: 824.966),

DOGS-NTA-Ni

(1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic

acid)succinyl] (nickel salt), MW: 1057.003) and DOGS-NTA (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1carboxypentyl)iminodiacetic acid)succinyl] (ammonium salt)) were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA). DOGS-NTA (ammonium salt) presented a molecular formula of C53H100N5O13 and a degree of purity>99%. It is guaranteed Nickel-free by the provider. Sphingomyelin (SM) from chicken egg yolk (≥95%) was purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). P19-4H (sequence: Cys-Ser-Arg-Ala-Arg-Lys-Gln-Ala-Ala-Ser-Ile-Lys-Val-Ala-Val-Ser-Ala-AspArg-His-His-His-His. (MW: 2428.7),60 AH peptide (sequence: Ser-Gly-Ser-Trp-Leu-Arg-Asp-Val-Trp-AspTrp-Ile-Cys-Thr-Val-Leu-Thr-Asp-Phe-Lys-Thr-Trp-Leu-Gln-Ser-Lys-Leu-Asp-Tyr-Lys-Asp,

MW:

3806.3)66,69 and T22, the antagonist peptide against CXCR4 (sequence : Arg-Arg-Trp-Cys-Tyr-Arg-LysCys-Tyr-Lys-Gly-Tyr-Cys-Tyr-Arg-Lys-Cys-Arg,

MW:

2488)80

were

synthesized

by

Genecust

(Luxembourg). Ultrapure (18.2 MΩ.cm) water was produced by PURELAB Option-Q (ELGA LabWater, Veolia Water STI, Antony, France). All the reagents were of the best analytical grade available.

2. Preparation of Large Unilamellar Vesicles (LUVs) Large Unilamellar Vesicles (LUVs) of different lipid compositions described in Table 1 were obtained. For this purpose, homogeneous mixtures of lipids including Phosphatidylcholine (PC), Phosphatidylserine (PS), Phosphatidylethanolamine (PE), Phosphatidylinositol 4,5-bisphosphate (PIP2), Sphingomyelin (SM), Phosphatidic Acid (PA) and Cholesterol (Chol) in various ratios (Table 1) were prepared in chloroform at room temperature. Chloroform was then removed under an argon stream until complete evaporation (at least 30 min). The phospholipidic film obtained on the wall of the flask was resuspended with a 20 mM HEPES, 100 mM NaCl, pH 7.4 buffer by thorough shaking to 8 ACS Paragon Plus Environment

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obtain MLVs (multilamellar vesicles) at a final concentration of 1 mg/mL. Six freeze-thaw cycles were then performed, and the subsequent LUVs were finally extruded (mini-extruder kit, Avanti Polar Lipid, Alabaster, Alabama, USA) through a 400 and a 100 nm pore size polycarbonate membrane successively, 21 times each. Dynamic Light Scattering measurements (Malvern Zetasizer Ver. 6.01, Malvern Instrument Ltd, UK) were conducted to determine the mean diameter of LUVs.

3. Cell-free expression of CXCR4 proteoliposomes. A homogeneous mixture of DOPC/DOPE/DMPA/Chol/DOGS-NTA in a 31:17:20:30:2 molar ratio (mol %) was prepared in chloroform (10 mg/mL) at room temperature. Chloroform was evaporated using a univapo 150H (Uniequip, Planegg, Germany). The thin lipid film obtained was rehydrated with 50 mM Tris buffer pH 7.5 to obtain a 30 mg/mL lipid mix. This solution was then sonicated at 20% (5 times during 30s) using a tip sonicator (Branson Digital Sonifier 250, Branson, Danbury, USA) before being filtered once with a 0.22 µm polyethersulfone membrane. N-terminal 6-Histidine-tagged CXCR4 gene sequence was cloned into a pIVEX2.4d (Roche Applied Science) using NdeI and XhoI restriction sites. Cell-free reaction was carried out at 30°C overnight at 600 rpm. E. coli extract and energy mix were provided by Synthelis SAS.73-74 To purify proteoliposomes, cell-free reactions were loaded on top of 3-step discontinuous sucrose gradient (60%, 30% and 5%) prepared in 50 mM Tris buffer pH 7.5. After ultracentrifugation at 200 000xg for 2h at 4°C, fractions were collected at each interface, 5-times diluted in 50 mM Tris buffer pH 7.5 and further centrifuged at 30 000xg for 30 min at 4°C. The resulting pellet was dissolved in a cryogenic buffer developed by Synthelis SAS. Negative proteoliposome controls (-) were prepared in the same experimental conditions at the same time as for CXCR4 proteoliposomes (+) with the same E. coli extract and energy mix but, without adding cDNA coding for CXCR4.

4. Characterization of CXCR4 proteoliposomes. CXCR4 proteoliposomes (+) and negative proteoliposome controls (-) were analyzed by Western blotting using an anti-poly-histidine antibody conjugated with a horseradish peroxidase (SigmaAldrich) diluted at 1/10 000 in Tris-buffered saline 0.1% Tween buffer, 5% nonfat milk. To assess to the protein and lipid concentrations in the proteoliposomes, they were subjected to acetone precipitation. Lipid concentration was assessed by Stewart assay81: lipids extracted in the organic phase after acetone precipitation of proteoliposomes were dried under a nitrogen stream and dissolved in chloroform. Then, 0.2 mL of lipids extracted in chloroform were used and, 1.8 mL of chloroform and 2 mL of ferrothiocyanate reagent at a final concentration of 247.5 mM were added. After centrifugation at 300 g during 10 min in glass tubes, the organic phase was collected, the absorbance was read at 485 nm and reported on a standard curve to assess the lipid concentration. 9 ACS Paragon Plus Environment

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Total protein concentration was measured by Bradford assay82 after dissolution in buffer (50 mM Tris buffer pH 7.5) of proteins obtained by acetone precipitation of proteoliposomes. CXCR4 concentration was estimated by Coomassie brilliant blue staining of an SDS-PAGE gel with the software ImageLab by comparing the intensity of CXCR4 band to the intensity of 3 different bands of increasing concentrations of bovine serum albumin (BSA) (relative quantification). Dynamic Light Scattering (DLS) measurements (Malvern Zetasizer Ver. 6.01, Malvern Instrument Ltd, UK) were conducted to determine the mean diameter of proteoliposomes.

5. Surface plasmon resonance imaging (SPRi). A Horiba SPRi+ biosensor (HORIBA Jobin Yvon SAS, Longjumeau, France) supplied with a bare gold (Au) SPRi-biochip (glass prism coated with a thin Au film) was used to monitor in real-time the formation of pep-tBLMs by SPRi. The surface of an Au biochip never exposed to air was cleaned with pure ethanol and then gently wiped with precision wipes tissue wipers (KIMTECH SCIENCE, VWR, US) before being thoroughly rinsed with ultrapure water (18.2 MΩ.cm) and dried under a filtered air stream. The cleaned biochip was then mounted on its support and put into the SPRi equipment. A poly(1,1,2,2-tetrafluoro)ethylene (Teflon) holed cylinder was used to delimitate directly on top of the bare gold a measurement area on the Au surface and a reaction chamber containing a 400 µL final buffer volume in which injections were made. All injections and rinsing steps were made manually by removing 100 µL of reactional medium and adding 100 µL of each solution or fresh buffer, in order to maintain constant the volume of the reaction chamber (400 µL). The experimental conditions used for the injection of P19-4H, which was the first step required for the formation of a pep-tBLM, were those previously optimized for P19 peptide, i.e. a concentration of 0.01 mg/mL and an incubation time of 45 min. Such conditions have been already demonstrated to lead to the formation of a homogeneous layer of the peptide on the gold surface.21 P19-4H peptide was first injected at a final concentration of 10 µg/mL, and LUVs were then added (at 100 µg/mL) before injection of AH peptide (at 200 µg/mL). Each injection was followed by a waiting time until signal stabilization before rinsing (12 times). The reflectivity changes were measured as a function of time. SPRi measurements were repeated 3 times. For each experience, the typical curve shown in the figures was the average of curves obtained from the measurements of 16 different and independent 500 µm diameter zones defined on the SPRi biochip.

6. NDPK-B binding assays Nucleoside diphosphate kinase-B (NDPK-B) (17 kDa) purified as previously described83 was kindly provided by Dr Thierry Granjon (ICBMS-UMR5246 Lyon, France). NDPK-B was injected in the SPRi chamber before and after formation of a DOPC/DOPS (75:25) pep-tBLM at a final concentration of 30 10 ACS Paragon Plus Environment

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nM, either in 20 mM Hepes buffer pH 7.4, supplemented or not with 150 mM NaCl. The reflectivity changes were measured as a function of time. After signal stabilization rinsing was performed to remove non-specific interactions. 7. Ligand binding assays on pep-tBLMs integrating CXCR4 P19-4H peptide was first injected (10 µg/mL) and then CXCR4 proteoliposomes or negative proteoliposome controls (with a minimal concentration of 100 µg/mL of lipids) with concomitant injection of 1 mM NiCl2 were incubated during 1h, before the final addition of AH peptide (200 µg/mL). The binding ligand capacity of CXCR4 was assessed with a synthetic antagonist, T22, at 1 µM before and after fusion of CXCR4-expressing proteoliposomes (+) or negative proteoliposome controls (-). Measurements were performed as described above. The amount of T22 bound was calculated according to formula given by the SPRi fabricant HORIBA and previously used to assess binding of peptides and small proteins using our instrument:84 ௠௔௦௦ ௔௥௘௔

=

௅zc∗∆ோ SP,R∗ఋ௡/ఋ௖

(1)

where ∆R is the reflectivity variation in percentage, LZC is the depth of penetration of the plasmon wave, here 1.02x10-4 mm, SP,R is the sensitivity of the SPR, here 2.25x103 %/refractive index unit (RIU) and δn/δc is the refractive index increment (mm3/pg). Here, δn/δc was considered to be 1.9 x 10-10 mm3/pg, which is an estimated average value for peptides and small proteins.85

8. Atomic Force Microscopy (AFM) A polycarbonate surface coated with a thin film of electrodeposited gold was used as a support. A PTFE tape was used to delimitate a surface large enough to contain a volume of 200 µL of 20 mM HEPES, 100 mM NaCl, pH 7.4 buffer. To characterize the formation of pep-tBLMs, successive injections of P19-4H (10 µg/mL), LUVs of 400 nm diameter (100 µg/mL) and AH peptide (200 µg/mL), and rinsing steps were performed under the same experimental conditions as for SPRi measurements. For imaging tethered proteoliposomes, P19-4H (10 µg/mL) and 25 µL of proteoliposomes were successively injected. AFM imaging during the different formation steps of pep-tBLMs or fixation of proteoliposomes was performed in liquid, using contact mode with a Solver PRO Scanning Probe Microscope, NT-MDT (Moscow, Russia) by scanning areas comprised between 5 x 5 µm2 and 50 x 50 µm2. Topographic images were acquired using silicon tips on integral cantilevers with a nominal spring constant of 3-130 mN/m and a frequency between 4 and 17 kHz. Representative images were acquired from ≥ 2 samples prepared on different days and from ≥ 3 macroscopically separated areas on each sample.


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9. Fluorescence Recovery After Photobleaching (FRAP) For

this

purpose,

the

fluorescent

probe

(18:1)

NBD-PE

(1,2-dioleyl-sn-glycero-3-

phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) was added into the LUVs at a 5% molar ratio. An inverted fluorescence microscope (Zeiss Observer.Z1, Le Pecq, France) equipped with a 20× objective was used to perform FRAP experiments. A circular zone of either liposome attached onto a gold surface via the P19-4H peptide or pep-tBLMs was photobleached by the mean of a laser beam at full power. The time used for photobleaching was typically 5 min for an area of 1.2.10-4 cm2. Then, the fluorescence recovery of the circle together with the surrounding area was regularly imaged at low laser power to avoid additional photobleaching41 and lateral diffusion coefficient of lipids was calculated with our homemade device by dividing the photobleached surface (cm2) by the time (seconds) needed to reach a total recovery of the zone. Each experiment was repeated 3 times. For FRAP measurements on tethered proteoliposomes and pep-tBLMs integrating CXCR4, a Laurdan-derivative, free fluorescent probe, kindly provided by Dr. Thierry Granjon (MEM2 team of ICBMS)86 and having a specific affinity for lipid bilayers was used with the same experimental conditions as described above.

III.

RESULTS

In order to develop a new approach allowing pep-tBLM formation with the possibility of modifying the lipid composition of the bilayer and adapting it for any (trans)membrane protein reinsertion, a new peptide tether has been designed. This peptide, called P19-4H (Figure 1C), was adapted from P19, previously used for pep-tBLM formation on gold surface.21,29,53-54,57 It has been modified by addition of four additional histidine residues at the C-terminal extremity.60 This polyhistidine region allows the fixation of any liposomes doped with a small amount of DOGS-NTA (2 molar % of the total lipid composition) by chelation in the presence of Nickel with a high binding affinity (Kd = 10−13 M for a six residue polyhistidine tag at pH 8.0),62 before vesicle disruption triggered by the fusogenic AH peptide and subsequent formation of the pep-tBLM (Figure 1A). As designed, this method leads to the formation of pep-tBLMs of variable lipid compositions, and thus, it can be adapted for the reconstitution of membrane proteins in any adequate lipid environment. As a proof of concept, pep-tBLMs of various compositions have been first characterized and the methodology transposed to the reinsertion of a functional membrane protein (Figure 1B). The transmembrane protein used as a model in this study was CXCR4, a 7 transmembrane domains GPCR synthesized by cell-free expression in proteoliposomes format embedding the protein in the lipid bilayer. The lipid composition of liposomes used to obtain cell-free expressed CXCR4 in proteoliposomes has been adapted to mimic the lipid environment required for a functional 12 ACS Paragon Plus Environment

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reinsertion of CXCR4, with a complex composition of 4 different lipids (DOPC/DOPE/DMPA/Chol, Table 1). The ligand binding capacity of CXCR4 was tested using T2280, a synthetic antagonist specific to this receptor, in order to check its functional folding and orientation in the bilayer.

Figure 1. (A) Principle of pep-tBLM formation using P19-4H, liposomes containing a chelating lipid, DOGS-NTA-Ni and AH 60

peptide

; (B) Principle of pep-tBLM integrating a membrane protein within the bilayer, using a cell-free expression

system (Synthelis SAS) ; (C) 3D modelling of P19-4H: the three characteristic parts of the peptide were represented with


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the cysteine residue in red for the grafting on the gold surface, the central α-helix in green for the spacing and the 4 Histidine residues in blue for the attachment of liposomes containing DOGS-NTA by chelation in the presence of Nickel salt. The sequence of P19-4H and P19 peptide from which P19-4H has been derived are indicated below. (D) Structure of the DOGS-NTA-Ni, a lipid with a chelating headgroup contained in liposomes, able to interact specifically with P19-4H.

1. pep-tBLM formation characterized by SPRi real-time monitoring The pep-tBLM formation of various compositions has been first characterized, step by step, by a SPRi real-time monitoring. SPRi is a label-free method based on an optical detection process that can sense any perturbation at the gold surface of the biochip, such as an interaction between probe molecules immobilized on the chip and captured target molecules, inducing a modification of resonance conditions at a constant angle, which are in turn seen as a change in reflectivity and which can be measured. Representative graphs were obtained from at least 3 independent experiments prepared on different days. Several lipid compositions were tested (Table 1), such as PC-containing liposomes, PS-containing liposomes, but also more complex mixtures such as natural lipid mixtures (egg PC/brain PS 68:32 or egg PC/brain PS/brain PIP2 68:30:2) to introduce a certain variability in the acyl chain composition, or cholesterol-containing compositions (DOPC/DOPE/DMPA/Chol 31:17:20:32), or a plasma membranemimicking composition (POPC/SM/POPE/Chol 44:35:10:11). The latest membrane composition permitted us to introduce, in addition to phospholipids, two other membrane lipid families sphingolipids and sterols.60

Table 1. Lipid compositions in molar percentage tested POPC (all doped with 2 % DOGS-NTA-Ni)

DOPC DOPC/DOPS (75:25) DOPC/DOPS doped with fluorophores Egg PC/brain PS (68:32) Egg PC/brain PS/brain PIP2 (68:30:2) DOPC/DOPE/DMPA/Chol (31:17:20:32) POPC/SM/POPE/Chol (44:35:10:11)


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All the compositions tested have given the same profile (Figure S1). For the sake of clarity, we only presented here typical results obtained during pep-tBLM formation, but each experiment was conducted three times for all the molar ratios investigated. Figure 2A shows a typical SPRi curve obtained during the formation of pep-tBLMs. It corresponds to the average of different measurements made from 16 independent 500 µm diameter zones defined on the SPRi-biochip (Figure 2A, inset). For all the lipid compositions tested, the same protocol has been followed: step 1) grafting of P19-4H peptide, step 2) fixation of LUV inserting 2% DOGS-NTA-Ni by metal-chelate interaction, step 3) fusion of liposomal membranes into pep-tBLMs triggered by a synthetic fusogenic peptide (AH) (Figure 1A). After injection of the P19-4H peptide (Figure 2A, arrow 1), we observed an instantaneous increase of the reflectivity percentage, followed by a stabilization of the signal to an approximate value of 5% of reflectivity after rinsing (r). This variation was characteristic of the attachment of the peptides to the gold biochip until saturation of the available surface. P19-4H interacts with the gold surface in a specific manner via its N-terminal cysteine residue that forms a covalent S-Au bond.16,87-88 The second step was the injection of LUVs (100 µg/mL) (Figure 2A, arrow 2), leading to a further increase of the percentage of reflectivity to a value around 25% after stabilization and rinsing. This step constituted the covalent attachment via chelation of the vesicles containing 2% DOGS-NTA-Ni to the four histidine residues of P19-4H available in C-terminal extremity. This type of bonding between the tether peptide like P19-4H and the chelating lipid like DOGS-NTA-Ni allowed us to modulate at will the lipid composition of the peptBLM, by changing the lipids contained in the tethered liposomes. In this way, the formation of the bilayer becomes independent from their lipid composition. Only 2% of Nickel-chelating head lipids (DOGS-NTA-Ni) were mandatory in the lipid composition of the vesicles. The third and final step for the formation of pep-tBLMs was the injection of AH peptide (Figure 2A, arrow 3) to induce the fusion of the tethered vesicles to obtain a planar tethered bilayer lipid membrane. After the injection, we observed a characteristic variation of the percentage of reflectivity, with a first increase due to the interaction of the peptide with the vesicles, followed by a decrease after induction of vesicle fusion. This decrease of reflectivity can be explained by the liposome fusion and the consecutive formation of a pep-tBLM. It can be stressed here that the fusion of our tethered liposomes by the AH peptide (Figure 2A) has given a similar SPRi reflectivity signal as the fusion of liposomes directly on a solid support to produce SLB.89 Final rinsing permitted elimination of residues (unbound membrane fragments…) and AH peptide as previously characterized.71-72 After signal stabilization, the value of reflectivity remained stable (at 13%). If the same experiments were performed without 2% DOGSNTA-Ni in the vesicles, unspecific interaction prohibited the AH peptide triggered fusion (Figure S2). The formation of bilayers using the AH peptide has been previously investigated by several methods including quartz crystal microbalance with dissipation monitoring or QCM-D,69 SPR,89 AFM90 and 15 ACS Paragon Plus Environment

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neutronic reflectometry.91 Hardy et al. showed that AH peptide could act on vesicles of complex lipid composition and that it was totally removed from reactional medium after vesicle fusion and rinsing. No trace of peptide has been found either on top of the bilayer or between the bilayer and the solid support.91 These findings suggested that AH peptide only bond curved vesicles but not planar membranes, and that it was removed from the lipid bilayer once the planar membrane was formed.71-72 In line with literature data, we observed no variation of the reflectivity if the AH peptide was reinjected on the formed bilayer. We used AFM to image the morphology of the surface at every final step of the formation of the pep-tBLM (Figure 2B) under similar experimental conditions as for SPRi investigations. Before P19-4H addition, a scratched surface due to the microstructure of the polycarbonate surface used, on which gold was electrodeposited, was observed (not shown). After incubation of tether peptides on top of the gold surface, we observed no changes neither in height nor in the surface morphology (Figure 2Ba, left). After liposome addition, a granulated surface with a mean approximate height of 50 nm corresponding to the tethered liposomes on P19-4H was obtained (Figure 2Bb, middle). Finally, after the addition of the fusogenic peptide, we saw a smooth homogeneous surface (Figure 2Bc, right) with a mean approximate height of 5 nm. This result was consistent with the previous SPRi results described above and confirmed the formation of a tethered bilayer lipid membrane on the gold surface via P19-4H peptide. All the compositions tested have given the same results. The methodology developed in this work allows the formation of pep-tBLMs of various compositions.


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Figure 2. Characterization of pep-tBLM formation by SPRi and AFM. (A) Formation of a pep-tBLM by fusion of tethered liposomes via a peptide spacer, on a thin layer of gold obtained by electrodeposition onto a polycarbonate plate. Typical SPRi curve showing the variation of percentage of reflectivity as a function of time. P19-4H was first injected at 10 µg/mL (arrow 1) leading to an increase of reflectivity to 5%, then after rinsing (r), liposomes of different phospholipid compositions including 2% of DOGS-NTA-Ni were added at 100 µg/mL, leading to a further increase of reflectivity to 25%. For this experiment, liposomes composed of DOPC/DOPS (75:25) +2% DOGS-NTA-Ni were used. After rinsing (r), the addition of AH peptide at 200 µg/mL (arrow 3) drove to the formation of a tethered bilayer shown by a first increase of the reflectivity percentage followed by a decrease of the reflectivity percentage. This experiment has been performed 3 times leading to the same typical curves. Same results have also been obtained for different compositions (see text; table 1). Inset: curves of the same experiment on 16 different and independent 500 µm diameter zones on the SPRi-biochip. The main curve shown in (A) is the average of these 16 independent measurements. The monitoring of the formation of a pep-tBLM by liposome fusion has been done 3 times with the same experimental conditions and led to the same results. (B) AFM topographic images (height) of gold electrodeposited on polycarbonate surface at the different steps of


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pep-tBLM formation described above. a) After P19-4H grafting: scratched surface due to the microstructure of the polycarbonate surface used, on which gold was electrodeposited; b) After tethering of liposomes composed of DOPC/DOPS (75:25) +2% DOGS-NTA-Ni: granulated surface; c) After AH peptide addition: smooth homogeneous surface after fusion. Same results have been obtained for different compositions (see text; table 1). Periodicity observed on AFM images is due to an optic artefact of the AFM set-up.

2. Formation of pep-tBLM integrating CXCR4 One of the main interests of using a tether peptide for the attachment of a lipid bilayer to a solid support is the possibility to insert membrane proteins with large ectodomains inside the membrane. The method used in this work for the insertion of our model protein CXCR4 in pep-tBLM consists in producing proteoliposomes embedding CXCR4 by a cell-free expression system (Synthelis SAS) and then, proceeding to the fusion of these proteoliposomes with the method described above.

a. Cell-free expression of CXCR4 in proteoliposome format We produced CXCR4 with a cell-free expression system allowing a one-step synthesis reaction of the protein with concomitant insertion in a lipid bilayer through the addition of liposomes to the reactional mix (Synthelis SAS). For this purpose, a liposome complex lipid composition for the cellfree reaction has been used to ensure the lipid requirement for functional insertion of CXCR4: DOPC/DOPE/DMPA/Chol, doped with 2% of DOGS-NTA, the chelating lipid allowing the fixation of the subsequent proteoliposomes on P19-4H. This composition has been previously shown to allow efficient cell-free protein synthesis of several membrane proteins in liposomes (VDAC, p7, NOX2/p22phox…)73-74,92 and it did not prevent the correct formation of the pep-tBLM, suggesting that we could really adapt the lipid composition of the bilayer to any (trans)membrane protein of interest, on the basis of the composition of the cellular membrane where this protein is expressed in a physiological or pathological context. This method leads to the formation of proteoliposomes embedding the protein within their membrane (Figure 3A). The quality of the synthesis reaction has been controlled by means of i) SDS-PAGE followed by a Coomassie Blue staining to estimate the quantity of CXCR4 and ii) western blotting to validate the full length protein and its overproduction. The yield of purified recombinant CXCR4 proteoliposomes was in the range of 50-250 µg/mL. The protein appeared on the gel around 37 kDa (Figure 3B, lanes 1 & 3). We can see also host cell proteins from E. coli lysate. The cell extract is like a “black box” in which numerous uncharacterized proteins are presents and remain after purification as shown in Figure 3B (lane 2) for the negative proteoliposome controls (-) prepared at the same time and under the same experimental conditions with the same cell extract and energy mix as for CXCR4 proteoliposomes (+), but without adding cDNA coding for CXCR4. 18 ACS Paragon Plus Environment

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The total protein concentration of proteoliposomes was constant and around 2000 µg/mL. The purity of CXCR4 in proteoliposomes was dependent on the expression yield and was therefore comprised between 10% and 50% of the total protein amount. The presence of DOGS-NTA, the chelating lipid, did not alter our cell-free production of CXCR4. Thus, we were able to produce sufficient amount of functional CXCR4. The total lipid concentration varied depending on the different production batches but was in the order of 1000 µg/mL. Proteoliposomes expressing CXCR4 (+) have a mean diameter of 567 nm with a polydispersity index of 0.284 while negative controls (-) have a mean diameter of 331 nm with a 0.166 polydispersity index (Figure S3 A & B). During the process of transcription and translation in the presence of liposomes, these latter fuse and reassemble to form larger vesicles, which explains the increase in the mean diameter of proteoliposomes expressing CXCR4 (+), in comparison with negative proteoliposome controls (-). The increase in the polydispersity index can be explained by the presence of proteoliposomes presenting variable quantities of host cell associated proteins.

Figure 3. (A) Scheme of the cell-free expression of CXCR4 in a proteoliposome format after addition of liposomes composed by DOPC/DOPE/DMPA/Chol/DOGS-NTA in a 31:17:20:30:2 molar ratio (mol %). (B) Images of a gel obtained after SDS-PAGE followed by Coomassie blue staining (1,2) and western blot with anti-Histidine antibody (1/10 000) (3,4) revealing the presence of overexpressed CXCR4 around 37 kDa in proteoliposomes (+) obtained by cell-free expression with addition of CXCR4 plasmidic DNA (1,3), and the absence of CXCR4 in negative proteoliposome controls (-) obtained by cell-free expression without addition of CXCR4 plasmidic DNA (2,4).

b. Proteoliposome fusion and subsequent pep-tBLM formation Experimental conditions for “empty” pep-tBLM formation described above were adapted for the fusion of proteoliposomes expressing CXCR4. The overexpressed protein CXCR4 was tagged in its Nterminal extremity by six histidine residues that were likely to interfere with the nickel salt contained in DOGS-NTA-Ni. Indeed, according to DLS measurements, proteoliposomes doped with DOGS-NTA19 ACS Paragon Plus Environment

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Ni aggregated (Figure S3 C), which could prevent the fusion of these vesicles tethered on P19-4H. To overcome this issue, DOGS-NTA-Ni was replaced in the lipid composition of proteoliposomes by a DOGS-NTA ammonium salt guaranteed Nickel-free (molecular formula: C53H100N5O13). Cell-free synthesis of CXCR4 proteoliposomes was therefore performed in the presence of the DOGS-NTA liposomes. The specific fixation by chelation of proteoliposomes to the tether peptide was then achieved by adding NiCl2 salt in the SPRi chamber. In the absence of Nickel, these proteoliposomes were able to bind to the surface after formation of the tether peptide layer, but they were not able to fuse after AH peptide addition (Figure S4). The optimal concentration and time of injection of the nickel salt was determined (data not shown). The best condition for this specific fixation was the concomitant injection of 1 mM NiCl2 with the proteoliposomes. SPRi profile obtained under these experimental conditions (Figure 4) was similar to that obtained in Figure 2 in the absence of transmembrane protein. The fixation of the tether peptide (Figure 4, arrow 1) induced an increase in the reflectivity. A more drastic increase was observed after injection of proteoliposomes with 1 mM NiCl2 (Figure 4, arrow 2), whereas the fusogenic peptide (Figure 4, arrow 3) injection led to a consequent decrease in the reflectivity signal. We noticed a larger decrease of reflectivity signal after the second rinsing step in the case of proteoliposome fixation than in case of the empty liposomes, which could be explained by a larger number of non-specific fixations of vesicles that were further eliminated by rinsing.


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Figure 4. SPRi real time monitoring of the formation of a pep-tBLM integrating CXCR4 by fusion of tethered proteoliposomes on a gold surface via a peptide spacer. The curve shows the variation of percentage of reflectivity as a function of time. P19-4H was first injected at 10 µg/mL (arrow 1) leading to an increase of reflectivity to 5%. Then, after rinsing (r), a volume of 50 µL of proteoliposomes were added (arrow 2), further increasing the reflectivity to 28%. After rinsing, the addition of AH peptide at 200 µg/mL (arrow 3) drove to the formation of a tethered bilayer integrating CXCR4. This was shown by a first increase followed by a reflectivity percentage decrease. Inset: curves of the same experiment on 16 different and independent 500 µm diameter zones on the SPRi-biochip. The main curve shown in the figure is the average of these 16 experiments. The monitoring of the formation of a pep-tBLM by proteoliposome fusion has been done 3 times with the same experimental conditions and led to the same results.

3. Characterization of pep-tBLM fluidity by FRAP measurements In order to check the lateral continuity of the (proteo)lipid bilayer obtained, we used fluorescence microscopy and FRAP measurements to study the dynamics of lipids inside the pep-tBLMs formed on a solid support. To this end, we added 5% of the fluorescent lipid NBD-PE to the lipid composition of the mixture to form fluorescent LUVs. FRAP experiences were performed after liposome fixation and after their fusion. For tethered liposomes (Figure 5A), we observed no recovery of the fluorescence in the photobleached area, even after 1h. This result was consistent with what is expected for attached vesicles in which the lipids cannot diffuse freely from one liposome to another.41 For fused liposomes (Figure 5, B & E), we observed a total fluorescence recovery after photobleaching within 8 min, with a diffusion coefficient (D) of 2.5.10-7 cm²/s. This value is of the same magnitude order of 21 ACS Paragon Plus Environment

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diffusion coefficients around 9-10.10-7 cm²/s recently determined by multiscale molecular dynamics simulation in POPC/POPS membrane models.93 Thus, we can conclude from this result that the peptBLM allows free lateral diffusion of the fluorescent probe within the bilayer, and that the tethered membrane is fluid and continuous. In order to check the lack of defects of the pep-tBLM (i.e. the presence of gold surface accessible, not recovered by a lipid bilayer), we took advantage of the peculiar adsorption properties of a cytoplasmic protein, Nucleoside Diphosphate Kinase – B (NDPKB). This protein is known to be able to associate with biological membranes containing anionic lipids such as PS in the absence of salts, this association being counteracted in the presence of a very high concentration of salts (150 mM NaCl).83,94 NDPK-B was first added in the SPRi chamber at a final concentration of 30 nM in the absence of a pep-tBLM. As shown in Figure 6A, NDPK-B readily adsorbed to the bare gold surface, as it induced a strong reflectivity increase both in the absence and in the presence of 150 mM NaCl. When injected on the top of a DOPC/DOPS (75:25) pep-tBLM (Figure 6B), NDPK-B induced a 5 % increase in the reflectivity in the absence of NaCl, attesting of protein binding to the lipid bilayer. No variation in the reflectivity was recorded in the presence of NaCl, confirming that the protein was not able to bind to the lipid bilayer (Figure 6B). The presence of a pep-tBLM thus completely abolished NDPK-B adsorption at the prism surface at 150 mM NaCl concentration, which confirmed us that the obtained lipid bilayer fully covered the surface thus impeding protein access to the gold surface. In the absence of NaCl, NDPK-B was able to bind the charged lipid PS present in the membrane. This result reinforces the conclusion that pep-tBLMs formed in our conditions were continuous, since any defect in the membrane covering the SPRi chip surface would have led to an unspecific interaction of NDPK-B on gold substrate. Finally, we tested as well the lipid dynamics of tethered proteoliposomes (Figure 5C) or peptBLMs integrating CXCR4 after fusion of the same proteoliposomes (Figure 5, D & F). We obtained a quite similar result as for pep-tBLMs without the protein, with the recovery of bleached area with a mobile fraction close to 100% within 10 min and a coefficient diffusion of 2.10-7 cm²/s for fused proteoliposomes. This coefficient diffusion value is consistent with diffusion coefficients around 23.10-7 cm²/s recently determined in more complex membrane compositions.95 All these results suggest that the presence of the protein within the membrane does not alter the mobility of the fluorophore in the pep-tBLMs.


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Figure 5. FRAP experiments on tethered liposomes or pep-tBLMs. (A) Typical images of tethered liposomes or (B) peptBLMs containing 5% of NBD-PE in their lipid composition. In the case of tethered liposomes (A), no recovery of -4

2

photobleaching was observed. For pep-tBLM (B), a total recovery of the photobleached area of 1.2.10 cm was obtained -7

2

in 8 min with a diffusion coefficient (D) of 2.5.10 cm /s. (C) Typical images of tethered proteoliposomes or (D) pep-tBLM integrating CXCR4 after addition of a free fluorescent probe (Laurdan derivative).

86

In the case of tethered

proteoliposomes (C), no recovery of the photobleached area was observed and for pep-tBLMs integrating CXCR4, we saw -7

2

a total recovery after 10 min with a diffusion coefficient D of 2.10 cm /s. (E) typical relative fluorescence intensity as a function of time curve for pep-tBLMs and (F) for pep-tBLM integrating CXCR4. This experiment was repeated 3 times leading to the same typical curves.


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Figure 6. SPRi monitoring of NDPK-B interaction with (A) a gold surface or (B) with a DOPC/DOPS (75:25) pep-tBLM (doped with 2% DOGS-NTA-Ni) in the absence (grey) or presence of 150 mM NaCl (brown). Protein injection was performed after formation of the pep-tBLM or directly on the gold surface (arrows).

4. Ligand binding assays on CXCR4 reinserted in pep-tBLMs In order to access to the functional folding of CXCR4 inserted in pep-tBLMs, we wanted to check the specific binding of the protein within the membrane with a ligand. For this purpose, we used a synthetic antagonist ligand, T22 ([Tyr5,12, Lys7]-polyphemusin II)80, that specifically inhibits human immunodeficiency virus type 1 (HIV-1) infection mediated by CXCR4. T22 binds to CXCR4 and interferes with stromal-cell-derived factor-1α (SDF-1α) – CXCR4 interactions in a competitive manner, SDF-1α being the natural ligand to CXCR4.96 As shown in Figure 7, the binding site of T22 involves i) the N-terminus extremity of CXCR4 and ii) two extracellular loops, one connecting the second (II) and the third (III) transmembrane domains, and the second connecting the fourth (IV) and the fifth (V) transmembrane domains (residues in purple). Therefore, the binding of T22 implies that CXCR4 must be both correctly inserted and correctly folded inside the bilayer with an appropriate spacing of the transmembrane domains. Additionally, the binding site of T22 is localised outside the membrane. Hence, characterising T22 binding is not only a good indicator of the functional folding of the receptor, but also gives information about the orientation of the receptor inside the membrane. Due to the presence of host cell proteins in proteoliposomes expressing CXCR4, we always performed binding experiments with negative proteoliposome controls (-), produced by cell-free system with the same cell extract and the same energy mix as the positive proteoliposomes (+), but without adding CXCR4 plasmid DNA. We injected the ligand before and after fusion of proteoliposomes, in order to not only confirm that the membrane receptor has kept its functional 24 ACS Paragon Plus Environment

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folding and a correct orientation both within tethered liposomes and pep-tBLMs, but also to estimate how much the fusion can lead to a loss of protein material. A higher increase of the reflectivity percentage was observed in Figure 8A after the addition of 1 µM of T22 subsequently to the tethering of CXCR4 proteoliposomes (+) compared to the addition of the same quantity of T22 after the fixation of negative proteoliposome controls (-) (Figure 8B). The unspecific interactions obtained with the negative proteoliposome controls (-) could be explained by the presence of host cell proteins in the liposomal membrane as shown in Figure 3B (lane 2). A significant final difference of reflectivity of 0.6% between proteoliposomes expressing CXCR4 (1.2 ± 0.3 %, Table 2, column 1) and negative proteoliposome controls (0.6 ± 0.1 %, Table 2, column 1) was obtained. The final amount of T22 bound has been calculated according equation (1) and the results were shown in Table 2 (column 2). The difference corresponds to a final amount of ~143 pg.mm-2. After fusion of proteoliposomes and T22 addition, a higher difference of reflectivity of 2.4% between pep-tBLMs expressing CXCR4 (Figure 8C & 4.5±0.5 %, Table 2, column 1) and negative controls (Figure 8D & 2.1

± 0.4%, Table 2, column 1) was recorded and corresponded to a final amount of ~573 pg.mm-2, suggesting that the loss of protein is limited after fusion of the vesicles. The kinetics from figure 8 were analysed and the initial binding rate was calculated for the 4 conditions tested by fitting the reflectivity (%) vs. time curve with a hyperbolic function (Table 2, columns 3 & 4). The binding rate was estimated at 3.7 ± 0.01 % of reflectivity.min-1 (corresponding to 840.65 ± 2.3 pg.mm-2.min-1) for T22 binding to CXCR4 inserted in surface-attached proteoliposomes and at 7.9 ± 0.01 % of reflectivity.min-1 for T22 binding (corresponding to 1896.84 ± 2.4 pg.mm-2.min-1) to CXCR4 inserted in the pep-tBLM bilayer. The non-specific absorption rate (negative proteoliposomes attached or fused) was estimated at 1.8 ± 0.01 %.min-1 (431.28 ± 2.4 pg.mm-2.min-1) and 4.7 ± 0.03 %.min-1 (1113.45 ± 7.1 pg.mm-2.min-1), respectively. All together, these ligand binding assays revealed that the reinsertion of a transmembrane protein in a reconstituted membrane attached to a grafted peptide spacer by a metal-chelate interaction before triggering vesicle fusion was functional. Additionally, the higher signal obtained for pep-tBLMs (after fusion) than for tethered-liposomes (before fusion) indicates that the protein remains correctly oriented and folded in the pep-tBLMs. Hence, the presence of 2% of chelating lipid in the liposomal membrane neither alter the production of an overexpressed membrane protein by cell free expression in proteoliposome format nor its functional orientation after pep-tBLM formation. This point can be certainly ascribed to the advantage that offers the new technology developed in this work to i) firstly produce a transmembrane protein in large amount by using an optimized cell free expression in an adequate lipid environment offered by the complex lipid composition of preformed liposomes that can be changed as will and, ii) to secondly use these preformed proteoliposomes to form pep-tBLMs by the way of a metal-chelate


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interaction which offers the versatility of grafting any liposomes of any lipid composition without affecting functionality of the inserted protein.

Figure 7. Scheme of CXCR4 structure when functionally inserted in the pep-tBLM on a gold surface. (A) Side view of the binding site of the antagonist ligand T22 with aminoacid residues involved in the interaction (highlighted in purple). (B) Top view of the receptor showing residues (purple) involved in T22 binding.


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Figure 8. SPRi monitoring of the interaction between T22, a CXCR4 synthetic antagonist at 1 µM (black arrow) and: (A) tethered CXCR4 proteoliposomes produced by cell-free expression; (B) tethered negative proteoliposome controls produced by cell-free expression without CXCR4 plasmid DNA; (C) pep-tBLM integrating CXCR4 after fusion of CXCR4 proteoliposomes by the AH peptide; (D) negative pep-tBLM control formed after fusion of negative proteoliposome controls produced by cell-free expression without CXCR4 plasmid DNA in the reactional mix. (E) Overlay of T22 binding curves on tethered CXCR4 proteoliposomes (+) (blue), tethered negative proteoliposome controls (-) (red), CXCR4 peptBLM (green) and negative pep-tBLM control (purple). We obtained a difference of reflectivity of 0.6% between tethered CXCR4 proteoliposomes (+) and negative proteoliposome control ones (-), a difference of 2.4% between pep-tBLM integrating CXCR4 and negative pep-tBLM control ones.


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Table 2. Final amount of T22 bound (pg.mm-2) and initial binding rate expressed in (%. min-1) or (pg.mm-2.min-1) estimated by SPRi measurements

Tethered CXCR4 proteoliposomes (+) Tethered Negative proteoliposome controls (-) Pep-tBLM integrating CXCR4 Negative control – pep-tBLMs

IV.

Average reflectivity increase after T22 addition and rinsing (%) 1.2 ± 0.3

Average T22 amount (pg.mm-2 )

Initial binding rate (%. min-1)

Initial binding rate (pg.mm-2.min-1)

286 ± 71

3.7 ± 0.01

840.65 ± 2.3

0.6 ± 0.1

143 ± 23

1.8 ± 0.01

431.28 ± 2.4

4.5 ± 0.5

1074 ± 119

7.9 ± 0.01

1896.84 ± 2.4

2.1 ± 0.4

501 ± 95

4.7 ± 0.03

1113.45 ± 7.1

DISCUSSION

Membrane proteins exhibiting extra- and intra-cellular domains which can extend to several tens of nanometers necessitate an adequate near-native lipid platform for their functional reconstitution. When reincorporated in SLBs, they suffer from non-physiological interactions with the solid support,4 leading to a loss of their dynamics and functionality.29-31 Consequently, it is essential to decouple the lipid bilayer from the supporting solid substrate to minimize interactions with the substrate, and to provide enough space for a functional folding of the transmembrane proteins. In this context, tBLMs, and especially pep-tBLMs manufactured on gold surface, have emerged as a very promising lipid matrix for functional membrane protein reconstitution.29 They combine mechanical stability and robustness of tethered bilayer lipid membranes guaranteed by the covalent attachment of the lipid bilayer onto a gold support, thus giving access to a manifold of surface-sensitive analytical techniques, such as quartz crystal microbalance (QCM-D),69 atomic force microscopy (AFM) or surface plasmon resonance imaging (SPRi);47-48 this latter offering the additional advantage of a real time label-free multiplexed detection for ligand binding assays and drug screening analysis.42 In tBLMs, the role of the tether/spacer molecules is to lift the lipid bilayer off the surface, and provide a hydrophilic layer between the lipid membrane and the substrate with, as far as possible, the functional properties of the cell environment. Spacers must establish a water-containing submembrane space, which reduces the influence of the metal surface, thus enabling the functional incorporation of membrane proteins.16 Peptide tethers have been shown to impart a satisfactory hydrophilicity and to offer anchored chains sufficiently apart, so as to accommodate water molecules and inorganic ions, and to create a suitable environment for the incorporation of integral proteins.55


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In this study, we developed a new technology enabling the formation of a lipid bilayer grafted onto an original peptide spacer (P19-4H)60 derived from the natural α-laminin thiopeptide (P19),29 by the way of a metal-chelate interaction (Figure 1). The main advantages of this technology are i) avoiding any chemistry to functionalize P19 and ii) suppressing the leaflet-by-leaflet formation of the bilayer as proposed in the classical way to form pep-tBLMs.30 Indeed, as mentioned in the introduction, pep-tBLMs are usually formed using a peptide spacer functionalized in their N-terminus by a sulfhydryl or lipoic acid group for self-assembly on gold, and activated in their C-terminus to covalently attach the amino group of dimyristoylphosphatidylethanolamine (DMPE).21,57 This thiolipopeptide forms the proximal layer by self-assembly on gold surface. The bilayer is subsequently obtained by vesicle fusion. In this topography, the formation of the bilayer in a twostep procedure could be unfavorable for reincorporation of integral membrane proteins which permanently require lipids for maintaining their function, and the phospholipid composition of the proximal leaflet of the bilayer cannot be varied. This latter point is critical and can restrict both the dynamic behavior of the tethered lipid bilayer and the capacity of the membrane protein to be reinserted in this latter. Our proposal is an alternative to the chemical methods. It is simplified since it allows the formation of the bilayer in a single step by triggering liposome fusion. In our approach, the membrane can be formed independently from the lipid composition of preformed liposomes, as long as a minor percentage of DOGS-NTA is present. Consequently, the lipid composition of the bilayer is the same as that of the tethered liposomes. This offers the possibility to change as will the lipid composition of both leaflets of the tethered membrane as demonstrated by the various lipid compositions we tested (Figures 2 & S1). Hence, it becomes possible to adapt the lipid composition of the bilayer to the membrane protein to incorporate to ensure a convenient lipid environment for its functional folding, as demonstrated for CXCR4 (Figures 4 & 8). By this way, we provide a biomimetic platform which is appropriate for functional insertion of polypeptides and proteins of different species and levels of complexity. Additionally, it has been reported that the formation of the distal leaflet on the preformed proximal one in the layer-by-layer procedure usually employed to prepare pep-tBLMs led sometimes to a heterogeneous and/or discontinuous tethered lipid bilayer due to the partial surface coverage with some non-disrupted liposomes adsorbed onto the solid surface.21,29,56 In the present study, AFM and FRAP characterizations allow us to show that pep-tBLMs inserting or not CXCR4 are fluid, homogeneous and continuous (Figures 2B & 5). The tethered bilayer formed onto P19-4H fully recovers the biochip and no gold surface is available after pep-tBLM formation (Figure 6). The values of the diffusion coefficients determined in presence or not of reinserted CXCR4 are 2.10-7 cm²/s or 2.5.10-7 cm²/s, respectively. These values are an order of magnitude higher than experimental ones, i.e. 2.10-8 cm²/s, reported in the literature for SLBs,19,41,97-98 even though experimental values of 29 ACS Paragon Plus Environment

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reported diffusion coefficients of both lipids and proteins vary widely, depending on the experimental technique used. However, the values determined in the present work correlate with the ones reported for coarse-grained simulations of complex asymmetric plasma membrane model.95 In simpler membrane models, the diffusion of the lipids in pure membranes, or with very few proteins, is around 9-10.10-7 cm²/s,93 while it decreases down to 4.10-7 cm²/s in highly protein crowded membranes.99 Fluorescence correlation spectrosocpy studies of ‘‘crowded’’ GUVs yielded lipid (DOPC/DOPG: 1.1.10-7 cm²/s) and protein (e.g. LacY: 0.4.10-7 cm²/s) diffusion coefficients in reasonable agreement with those above.100 These results suggest that the lateral diffusion in peptBLMs formed onto P19-4H is most likely nearer to that of the plasma membrane than for SLBs. This difference can be ascribed to the detaching of the membrane from the surface and suggests that the membrane in pep-tBLMs formed onto P19-4H is lifted off enough to suppress any influence of the metal surface or unfavorable frictions on the gold support. Finally, we succeeded in reinserting CXCR4 (C-X-C motif chemokine receptor 4), a seventransmembrane protein belonging to the large superfamily of G-protein-coupled receptors (GPCRs), in pep-tBLMs of adapted lipid composition, formed on P19-4H by fusion of proteoliposomes obtained by functional cell-free expression of CXCR4 (Figure 4). Ligand binding assays have revealed that the integrated receptor presents both a functional folding and a functional orientation (Figures 7 & 8). As mentioned in the introduction, several membrane proteins have been successfully reincorporated in pep-tBLMs,21,36,49-51,53-55,61 but only few studies have investigated the functionality or the activity of the reinserted membrane protein in tBLMs.22,57 A possible explanation of this failure was the small amount of reincorporated proteins.53 In our approach, the pep-tBLMs have been prepared using proteoliposomes produced by an efficient cell-free protein expression system, in which the complex lipid composition has been adapted to the over-expressed protein73-76 and the liposomal membranes enriched in the protein of interest, CXCR4 (with concentrations varying between 200-1000 µg/mL, Figure 3), in spite of the presence of a small fraction of the chelating lipid. We believe that under such conditions, i) the amount of CXCR4 functionally folded in the proteoliposome membranes is large enough to enrich pep-tBLMs even after fusion of proteoliposomes and substantial loss of proteic materials, and ii) that protein denaturation during pep-tBLM formation is restricted, compared to the usual layer-by-layer process for obtaining pep-tBLMs. In the end, compared with tBLMs produced by vesicle fusion triggering with AH peptide onto a gold surface using phospholipidpolyethylene glycol (PEG) as a spacer, our system presents a last advantage to not present any polymer chains at the upper side of the lipid bilayer, which can prevent any ligand recognition.23


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V.

CONCLUSION

The perspective of producing membrane proteins and integrating them in a functional folding and orientation at the surface of a sensor chip is a technological challenge of great interest for fundamental researches on biological membranes and for many applications in ligand binding study and drug screening. In this article, we provide a new technology for the achievement of a new generation of pep-tBMs as a promising lipid membrane platform which facilitates functional incorporation of transmembrane proteins in an optimal lipid environment on a gold support, an electrically conducting substrate which facilitates investigation by various techniques such as quartz crystal microbalance, electrochemistry, or surface plasmon resonance imaging, well adapted to develop membrane biochips for drug screening assays. Based on a metal-chelate interaction between an especially designed peptide derived from natural thiopeptide, P19-4H, and the chelating lipid headgroup of DOGS-NTA inserted in a small fraction in preformed (proteo)liposomes before triggering vesicle fusion, this new technology offers the possibilities i) to change at will the lipid composition of the pep-tBLMs and, hence, ii) to adapt the lipid bilayer composition of the tethered membrane to each transmembrane protein that we want to reincorporate. Thus, the lipid composition does no longer represent a limitation for obtaining a proteo-lipid planar bilayer. This advantage constitutes a guarantee that the protein will be reinserted in a native-like lipid environment, which is a crucial criterion to keep it in a natural folding to maintain its biological activity on a gold surface to properly evaluate their functionality. In addition, the conformation, length and hydrophilic properties of the tether peptide may be controlled by the nature and number of residues of amino acids. Therefore, it should be possible to adjust the sequence of the tether peptide according to space and physico-chemical conditions needed for the reinserted membrane protein ectodomains. At the end, we can also foresee to use a peptide mimicking ultimately the structure of the extracellular matrix, in order to improve the environment adaptation to the expressed protein in a more extended way. All these considerations make the present pep-tBLM construction process a method of choice for the development of transmembrane protein biochips.

SUPPORTING INFORMATION Figure S1: SPRi monitoring of the formation of pep-tBLM by fusion of tethered liposomes of various compositions on a gold surface via a peptide spacer. Figure S2: Negative controls for pep-tBLM formation. Figure S3: Graphs of the size distribution by intensity of proteoliposomes expressing his-tagged CXCR4 or negative proteoliposome controls characterized by Dynamic Light Scattering (DLS). Figure S4: Negative controls for pep-tBLM formation integrating CXCR4. 31 ACS Paragon Plus Environment

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AUTHOR INFORMATION Corresponding author *Email: [email protected]

ACKNOWLEDGMENTS We would like to thank Dr Thierry Granjon for providing the fluorescent Laurdan-like probe for FRAP measurements and the purified Nucleoside diphosphate kinase-B (NDPK-B). This work was supported by a CIFRE (Conventions Industrielles de Formation par la REcherche) convention, number 2014/0745, of the ANRT (Association Nationale de la Recherche et de la Technologie) and Synthelis SAS. DLS measurements were performed at CarMeN laboratory (team 4), IMBL (Villeurbanne, France). REFERENCES (1) Pierce, K. L.; Premont, R. T.; Lefkowitz, R. J. Seven-transmembrane receptors. Nat. Rev. Mol. Cell Biol. 2002, 3 (9), 639-650. (2) Tamm, L. K.; McConnell, H. M. Supported phospholipid bilayers. Biophys. J. 1985, 47 (1), 105-113. (3) Brian, A. A.; McConnell, H. M. Allogeneic stimulation of cytotoxic T cells by supported planar membranes. Proc. Natl. Acad. Sci. USA 1984, 81 (19), 6159-6163. (4) Wagner, M. L.; Tamm, L. K. Tethered Polymer-Supported Planar Lipid Bilayers for Reconstitution of Integral Membrane Proteins: Silane-Polyethyleneglycol-Lipid as a Cushion and Covalent Linker. Biophys. J. 2000, 79 (3), 1400-1414. (5) Sackmann, E. Supported Membranes: Scientific and Practical Applications. Science 1996, 271 (5245), 43-48. (6) Sackmann, E.; Tanaka, M. Supported membranes on soft polymer cushions: fabrication, characterization and applications. Trends Biotechnol. 2000, 18 (2), 58-64. (7) Silin, V. I.; Wieder, H.; Woodward, J. T.; Valincius, G.; Offenhausser, A.; Plant, A. L. The Role of Surface Free Energy on the Formation of Hybrid Bilayer Membranes. J. Am. Chem. Soc. 2002, 124 (49), 14676-14683. (8) Terrettaz, S.; Mayer, M.; Vogel, H. Highly Electrically Insulating Tethered Lipid Bilayers for Probing the Function of Ion Channel Proteins. Langmuir 2003, 19 (14), 5567-5569. (9) Cullison, J. K.; Hawkridge, F. M.; Nakashima, N.; Yoshikawa, S. A Study of Cytochrome c Oxidase in Lipid Bilayer Membranes on Electrode Surfaces. Langmuir 1994, 10 (3), 877-882. (10) Ogier, S. D.; Bushby, R. J.; Cheng, Y.; Evans, S. D.; Evans, S. W.; Jenkins, A. T. A.; Knowles, P. F.; Miles, R. E. Suspended Planar Phospholipid Bilayers on Micromachined Supports. Langmuir 2000, 16 (13), 5696-5701. (11) Römer, W.; Steinem, C. Impedance Analysis and Single-Channel Recordings on Nano-Black Lipid Membranes Based on Porous Alumina. Biophys. J. 2004, 86 (2), 955-965. (12) Römer, W.; Lam, Y. H.; Fischer, D.; Watts, A.; Fischer, W. B.; Göring, P.; Wehrspohn, R. B.; Gösele, U.; Steinem, C. Channel Activity of a Viral Transmembrane Peptide in Micro-BLMs: Vpu1-32 from HIV-1. J. Am. Chem. Soc. 2004, 126 (49), 16267-16274. (13) Guidelli, R.; Aloisi, G.; Becucci, L.; Dolfi, A.; Rosa Moncelli, M.; Tadini Buoninsegni, F. Bioelectrochemistry at metal/water interfaces. J. Electroanal. Chem. 2001, 504 (1), 1-28. (14) Naumann, R.; Schiller, S. M.; Giess, F.; Grohe, B.; Hartman, K. B.; Kärcher, I.; Köper, I.; Lübben, J.; Vasilev, K.; Knoll, W. Tethered Lipid Bilayers on Ultraflat Gold Surfaces. Langmuir 2003, 19 (13), 54355443. 32 ACS Paragon Plus Environment

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