Histidine-Tagged Amphiphiles for the Reversible Formation of Lipid

Langmuir 2000, 16, 5471-5478

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Histidine-Tagged Amphiphiles for the Reversible Formation of Lipid Bilayer Aggregates on Chelator-Functionalized Gold Surfaces Thierry Stora,† Zoltan Dienes,† Horst Vogel,* and Claus Duschl* Laboratoire de chimie physique des polyme` res et membranes, Ecole Polytechnique Fe´ de´ rale de Lausanne, CH-1015 Lausanne, Switzerland Received December 31, 1999. In Final Form: March 17, 2000 A new strategy for the reversible formation of supported lipid bilayers is introduced. It uses an extension of immobilized metal ion affinity chromatography technology, which depends on the reversible formation of complexes between metal ion binding chelator groups and oligohistidines. Lipid layers containing hexahistidine-derivatized amphiphiles (His-lipids) are anchored to a self-assembled monolayer (SAM) containing synthetic chelator thioalkanes (CTA). The control over the density of anchor sites on the substrate and over the surface wetting properties leads to a high degree of flexibility in the design of extra free space between the substrate and the lipid layer for the reconstitution of proteins. His-lipids can be used to anchor planar lipid layers or layers of intact lipid vesicles to CTA SAMs. The form of the lipid layers (planar bilayer or vesicles) is determined by the charge on the lipids and the ionic strength of the buffer solution used. Surface plasmon resonance is used to monitor the formation of lipid layers. The synthesis of the His-lipid and the characterization of its monolayer layer forming and binding properties on a Langmuir trough are also described.

Introduction Transmembrane receptor proteins, which upon binding of ligand molecules act as ion channels or trigger the catalysis of secondary reaction (e.g., activation of Gproteins), are currently intensively studied.1 The investigation of the function-structure relationship of these proteins may offer new insight into cellular processes occurring in the vicinity of membranes and may also be used for the development of new analytical devices for pharmaceutical evaluation.2-6 A prerequisite to this is the reconstitution of the fragile membrane-spanning proteins under defined conditions. The presence of an amphiphilic environment is essential for these proteins to preserve their integrity and consequently their function. Micelles, lipid vesicles, or planar lipid bilayers are examples of artificial systems that serve these requirements. The formation of such lipid aggregates at solid-fluid interfaces offers a number of favorable features:7 first, the asymmetric environment offers a simple way for prealigning proteins; second, the surface allows molecules to be anchored to it enabling the buildup of mechanically stable systems; third, the availability of a range of new highly specific analytical surface sensitive techniques gives access to the properties of these systems on a molecular level. The major disadvantage of this approach is that the * To whom correspondence should be addressed. E-mail: Horst. [email protected], [email protected]. † T.S. and Z.D. are now with Firmenich SA, Geneva, Switzerland. (1) Heyse, S.; Stora, T.; Schmid, E.; Lakey, J. H.; Vogel, H. Biochim. Biophys. Acta 1998, 1376, 319. (2) Bieri, C.; Ernst, O. P.; Heyse, S.; Hofmann, K. P.; Vogel, H. Nat. Biotechnol. 1999, 17, 1105. (3) Cornell, B. A.; Braach-Maksvytis, V.; King, L. G.; Osman, P. D. J.; Raguse., B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580. (4) Gritsch, S.; Nollert, P.; Ja¨hnig, F.; Sackmann, E. Langmuir 1998, 14, 3118. (5) Kro¨ger, D.; Hucho, F.; Vogel, H. Anal. Chem. 1999, 71, 3157. (6) Stora, T.; Lakey, J. H.; Vogel, H. Angew. Chem., Int. Ed. Engl. 1999, 38, 389. (7) Sackmann, E. Sciene 1996, 271, 43.

close proximity between the hard substrate surface and the membrane can cause strong nonspecific coupling that affects the properties of the latter and consequently any protein therein. Several strategies to form supported lipid bilayers for the reconstitution of proteins have already been successfully introduced. Physisorbed lipid bilayers on oxide surfaces,8,9 on functionalized self-assembled monolayers10 or on polymer cushions11 on one hand and planar lipid bilayers that are anchored covalently to the substrate via sulfur-bearing amphiphiles,12-14 peptide spacers,15 or alkyl-chain-derivatized polymers16 on the other hand represent the most promising examples so far. We present a new strategy based on the use of histidinederivatized lipids (named His-lipids in the following) for the reversible formation of supported lipid layers on chelator-functionalized gold surfaces either as planar lipid bilayers or as layers of intact lipid vesicles. The anchoring of the lipid layer is an extension to immobilized metal affinity chromatography (IMAC),17,18 which is broadly used for the purification of recombinant proteins bearing a polyhistidine sequence. Chelators immobilized on a chromatography resin form complexes with transition metal (8) Johnson, S. J.; Bayerl, T. M.; McDermott, D. C.; Adam, G. W.; Rennie, A. R.; Thomas, R. K.; Sackmann, E. Biophys. J. 1991, 289, 289. (9) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307. (10) Duschl, C.; Liley, M.; Corradin, G.; Vogel, H. Biophys. J. 1994, 67, 1229. (11) Ku¨hner, M.; Tampe´, R.; Sackmann, E. Biophys. J. 1994, 67, 217. (12) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197. (13) Heyse, S.; Vogel, H.; Sa¨nger, M.; Sigrist, H. Protein Sci. 1995, 4, 2532. (14) Williams, L. M.; Evans, S. D.; Flynn, T. M.; Marsh, A.; Knowles, P. F.; Bushby, R. J.; Boden, N. Langmuir 1997, 13, 751. (15) Naumann, R.; Jonczyk, A.; Kopp, R.; Esch, J. v.; Ringsdorf, H.; Knoll, W.; Gra¨ber, P. Angew. Chem., Int. Ed. Engl. 1995, 34, 2056. (16) Spinke, J.; Yang, J.; Liley, M.; Ringsdorf, H.; Knoll, W. Biophys. J. 1992, 63, 1667. (17) Porath, J.; Carlsson, J.; Olsson, I.; Belfrage, G. Nature 1975, 258, 598. (18) Hochuli, E. In Genetic Engineering; Setlow, J. K., Ed.; Plenum Press: New York, 1990; Vol. 12, p 87.

10.1021/la991711h CCC: $19.00 © 2000 American Chemical Society Published on Web 05/16/2000

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Langmuir, Vol. 16, No. 12, 2000

Stora et al.

ions. The free coordination sites are subsequently filled by additional electron-donating groups such as histidine residues. The protein adsorption is fully reversible either on addition of a competitive ligand (histidine, imidazole), protonation of the histidine, or removal of the metal ion via EDTA complexation. Binding of histidine-containing peptides19 and proteins20 to chelator-lipid monolayers at the air/water interface has been demonstrated in the past. Recently, we have applied this method using a synthetic chelator thioalkane (CTA), which can form self-assembled monolayers (SAMs) on gold surfaces via its thiol group. In such SAMs, nitrilotriacetic acid (NTA) groups are exposed that chelate transition metal ions. Reversible and oriented binding of Fab fragments modified with a C-terminal hexahistidine extension was monitored in situ using surface plasmon resonance.21 The His-lipid we synthesized for anchoring the lipid bilayers is a dioctadecylamine (DODA) with a histidine extension coupled to it via an ethylene glycol spacer. The use of hydrophilic spacer units ensures a high degree of flexibility and the creation of sufficient hydrated space between the lipid layer and the substrate for the accommodation of extramembranous parts of the proteins. These His-lipids can be introduced into micelles or vesicles in controlled amounts by simply mixing them to any desired lipid composition or eventually by exchange processes to cell fragments or whole cells. In this paper we describe the synthesis of the His-lipid and present its thermodynamic characterization and its binding properties to an NTA-conjugated rhodamine chromophore using a Langmuir film balance. The formation of His-lipid-containing planar bilayers on NTAfunctionalized SAMs from detergent and vesicle solutions is investigated in detail. Both the content of His-lipid in the dispersion and the concentration of CTA in the SAMs is systematically varied. Through the addition of charged lipids to the vesicles in low ionic strength buffer, we could observe reversible adsorption of intact vesicles (see Figure 1). We used surface plasmon resonance (SPR)22 for the layer characterization and for the in situ monitoring of the layer formation at the gold/buffer interface. Its high sensitivity enables a quantitative determination of the amount of adsorbed molecules per unit area. Experimental Section Materials and General Methods. Reagents were purchased from Fluka at the highest available quality and used as supplied. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-Palmitoyl-2-oleoyl-sn-glycero-3-phospho-1′-glycerol (POPG) was obtained from Avanti Polar Lipids (Birmingham, AL). N-(7Nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dimyristoyl-sn-glycero-3phosphoethanolamine (NBD-PE) was obtained from Molecular Probes (Eugene, OR). NR,NR-Bis(carboxymethyl)-Nω-(11-mercaptoundecanoyl-glycyl-glycyl-glycyl)-L-lysine (CTA for chelator thioalkane) was synthesized as described elsewhere.21 Resinbound NH2-His6 was obtained from the Institut de Biochimie, University of Lausanne. Experiments were performed in HPLCgrade solvents or in ultrapure water (NANOpure quality 18 MΩ cm). Triethylamine and diisopropylethylamine were distilled over KOH. NMR spectra were measured with a Bruker AC-200 spectrometer at 200 and 50 MHz for 1H and 13C, respectively. Mass spectra (MS) were recorded on a Finnigan Mat SSQ 710C spectrometer by electron spray ionization (ESI). Thin-layer (19) Dietrich, C.; Schmitt, L.; Tampe´, R. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 9014. (20) Ng, K.; Pack, D. W.; Sasaki, D. Y.; Arnold, F. H. Langmuir 1995, 11, 4048. (21) Keller, T. A.; Duschl, C.; Kro¨ger, D.; Se´vin-Landais, A.-F.; Cervigni, S. E.; Dumy, P.; Vogel, H. Supramol. Sci. 1995, 2, 155. (22) Knoll, W. MRS Bulletin 1991, XVI, 29.

Figure 1. Scheme of supported lipid bilayer aggregates containing DODA-EG3-His6 lipid on a NTA-functionalized SAM. chromatography was carried out on precoated plates (0.2 mm Silica Gel 60 F 254 on aluminum support). Ce-Mo sulfate was used for visualization of the products. 11-Dioctadecylamido-3,6,9-trioxaundecanoic acid (DODAEG3). 3,6,9-Trioxaundecanedioic acid (1.11 g, 5 mmol) was dissolved in 40 mL of CH2Cl2, and N,N′-dicyclohexylcarbodiimide (DCCI) (1.24 g, 6 mmol, in 20 mL of CH2Cl2) was slowly added in portions and vigorously stirred for 12 h at 20 °C. The N′,NDicyclohexylurea (DCU) formed was filtered off, and the solution was concentrated to about 5 mL through evaporation of excess solvent. It was then added to a solution of DODA (522 mg, 1 mmol) and N,N-diisopropylethylamine (DIEA) (0.34 mL) in 5 mL of CHCl3:iPrOH 4:1. After 24 h stirring at 20 °C, the solution was washed with 1 M HCl and brine and dried over Na2SO4. The product was purified by flash chromatography (Silica Gel 60, CHCl3:MeOH:H2O 80:18:2, Rf ) 0.57). Yield: 500 mg (70%) lipid. 1H NMR (CDCl :CD OD 4:1): δ 4.29, 3.98 (2s, 4H, H-C(2,10)); 3 3 3.76, 3.70 (2s, 8H, H-C(4,5,7,8)); 3.29, 3.13 (2t, J ) 7.5, 4H, N-CH2-); 1.53 (m, 4H, -CH2CH2N); 1.26 (m, 60H, fatty acid); 0.88 (t, J ) 6.5, 6H, CH3). 13C NMR (CDCl3:CD3OD 4:1): δ 175.8

Histidine-Tagged Amphiphiles (COO); 169.8 (CON); 77.7, 70.8, 70.0, 69.8, 69.6, 69.2 (-CH2O); 47.2; 46.5 (N-CH2); 32.1, 29.9, 29.6, 28.8, 27.7, 27.3, 27.1, 22.9, 14.2 (fatty acid). MS (ESI): 728.0. C44H87NO6 (726.2). NR-(11-Dioctadecylamido-3,6,9-trioxaundecanedioyl)pentahistidylhistidine (DODA-EG3-His6). DODA-EG3 (35 mg, 48 µmol), N-hydroxysuccinimide (7 mg, 60 µmol, in 30 µL of DMF), and DCCI (12 mg, 58 µmol) was dissolved in 1 mL of CH2Cl2, and then the mixture was stirred for 5 h at 20 °C. DCU was filtered off, and the solution was added to NH2-His6-resin (approximately 14 µmol as determined by cleavage and His6 recovery) in 1 mL of DMF with 40 µL of DIEA. After stirring overnight at 20 °C, the resin was filtered off and washed with CHCl3-MeOH 5:1. It was then treated with 1 mL of the cleavage mixture (trifluoroacetic acid (TFA):Et3SiH:H2O 92.5:5.0:2.5) for 2.5 h at 20°. After filtration, drying, cold Et2O precipitation, and decanting, the product was redissolved in CHCl3, washed with buffer (10 mM Na-EDTA, 10 mM NaiP, pH ) 7.4), and dried over Na2SO4. Yield: 7 mg lipid. 1H NMR (CD3OD): δ 8.86 7.42 (2m, 12H, H-imidazole); 4.70 (m, 6H, H-CR-His); 4.37, 4.03 (2s, 4H, H-C(2,10)); 3.8-3.6 (m, 8H, H-C(4,5,7,8)); 3.4-3.1 (m, 16H, H-His, N-CH2-); 1.61 (m, 4H, -CH2CH2N); 1.32 (m, 60H, fatty acid); 0.94 (t, J ) 6.5, 6H, CH3). MS (ESI): 1550.3. C80H129N19O12Na (1548.6). NR,NR-Bis(carboxymethyl)-Nω-(rhodamine-3/4-thiocarbamoyl)L-lysine (Rho-NTA). NR,NR-Bis(carboxymethyl)-L-lysine tris-(triethylamine) salt (28 mg, 50 µmol) and rhodamine-3/4-isothiocyanate (27 mg, 50 µmol) was suspended in 2 mL of methanol with 40 µL of triethylamine. After 1 h stirring in the dark at 20 °C, the solvent was evaporated and the residue was acidified with 1 M HCl. The product was purified by flash chromatography (Silica 60, in the dark, acetonitrile:water 9:1, Rf ) 0.4). Yield: 12 mg (30%) red solid. 1H NMR (D2O): δ 8.0-6.5 (m, 9Hrhodamine); 3.75, 3.70 (2s, 4H, H-C(2′); 3.6-3.3 (m, 3H, H-C(2,6)); 3.15 (q, 8H, Et); 1.84 (m, 2H, H-C(3)); 1.20 (t, 12H, Et); 1.10 (m, 4H, H-C(4,5)). MS (ESI): 763.0, 575.4. C39H48N5O9S (762.8). Film Balance Measurements. Experiments were carried out on a homemade Teflon Langmuir trough (25 mm × 130 mm, about 10 mL subphase volume) placed on a commercial frame comprising two symmetrically moveable barriers (Riegler & Kirstein, Germany). Pressure-area diagrams were obtained by isothermal compression and expansion. The surface tension was measured with a Wilhelmy system at 20 °C. Subphase exchange was performed with an Ismatec peristaltic pump (1 mL/min) with a needle input and output installed behind the barriers. Reagents were also introduced through these needles without disturbing the air-water interface. The trough was mounted on a three-directional, motor-stepper- driven translation stage of a Zeiss Axiotron epifluorescence microscope. The filter sets used correspond to NBD fluorescence (excitation at 450-490 nm, emission at 515-565 nm) and rhodamine fluorescence (excitation at 546 nm, emission at 590 nm). The images were recorded with a Hamamatsu SIT video camera attached to the microscope. For monolayer experiments, 10 µL (0.7 mM in chloroform) lipid solutions were spread on 10 mM Na-phosphate subphase at pH 7.4. For binding measurements, 10 mL (0.45 mM total concentration) lipid mixtures (90% DMPE, 9% DODA-EG3-His6, 1% NBD-PE) were spread on 10 mM Na-phosphate/250 mM NaCl subphase at pH 7.4 and compressed to 18.5 mN/m. Rho-NTA (5 mM) with or without 20 mM NiCl2 in the same buffer was introduced via the installed needle. Subphase exchange was carried out with the same buffer containing 0.1 mM Na-EDTA or 50 mM imidazole. Surface Plasmon Resonance Spectroscopy. The surface plasmon resonance measurements were performed, using the Kretschmann coupling scheme,23 on a home-built computerized reflection apparatus as described in detail elsewhere.24 At the resonance angle, the incident laser beam (He-Ne laser, 632.8 nm, p-polarized) couples via an equilateral, high refractive index prism (SF 10, n ) 1.723) to the surface plasmon mode in a thin gold film. The optical properties of the thin organic film were derived by fitting the experimental reflectivity vs angle (θ-2θ) scans with curves calculated according to the Fresnel equations. (23) Kretschmann, E. Opt. Commun. 1972, 6, 185. (24) Terrettaz, S.; Stora, T.; Duschl, C.; Vogel, H. Langmuir 1993, 9, 1361.

Langmuir, Vol. 16, No. 12, 2000 5473 For the determination of the thicknesses of the lipid layers a refractive index n ) 1.45 was used. The time-dependent measurements were taken at a fixed angle, slightly smaller than the resonance angle, at the steepest part of the resonance curve. Layer Formation. Self-assembly of CTA (0.1-0.2 mg/mL, 0.15-0.3 mM) was performed for 4-12 h in water containing 1% TFA. TFA improves the solubility of CTA in aqueous buffers. After extensive water and buffer (sodium phosphate with 10 mM or 250 mM NaCl, pH ) 7.4) wash followed by reequilibration in water, Ni2+ was added (50 mM in water) for 15 min. The surface was again washed with water and then buffer. The selfassembly experiments of the lipid layers were either performed in deionized water (18 MΩ cm) or sodium phosphate buffer containing 10 or 250 mM NaCl at pH ) 7.4. The surface was incubated with DODA-EG3-His6 (His-lipid) solution (0.2 mg/ mL) in n-octyl-β-glucoside (OG) or sonicated vesicle solutions (0.04-0.4 mg/mL in buffer). Each incubation was followed by a washing step in the respective buffer or solvent. After experiments, the CTA sublayer was restored by extensive imidazole (500 mM in buffer, pH ) 7.9), EDTA (10 mM in buffer), and OG (48 mM in buffer) wash. Vesicle Preparation. The lipids (POPC, His-lipid, and in some of the experiments negatively charged POPG) were mixed in the desired molar composition (typically 1 mg overall weight) in a glass vial from chloroform solution. The solvent was evaporated under a nitrogen flow. Buffer was (50 µL) added to the lipid, and the resultant lipid dispersion was transferred to a 1 mL sized Eppendorf vial and sonicated at constant temperature in a bath sonifier for typically 15 min. A clear solution was obtained containing small unilamellar vesicles (SUVs) with a diameter