Langmuir 2007, 23, 4303-4309
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Free-Standing Films of Fluorinated Surfactants as 2D Matrices for Organizing Detergent-Solubilized Membrane Proteins Vera Petkova,† Jean-Jacques Benattar,† Manuela Zoonens,‡,§ Francesca Zito,‡ Jean-Luc Popot,‡ Ange Polidori,| Sylvain Jasseron,| and Bernard Pucci*,| SerVice de Physique de l’Etat Condense´ , CEA-Saclay, DSM/DRECAM, F-91191 Gif sur YVette, France, UMR 7099, CNRS & UniVersite´ Paris 7, Institut de Biologie Physico-Chimique, CNRS FRC 550, 13 rue Pierre et Marie Curie, F-75005 Paris, France, and UniVersite´ d’AVignon, Laboratoire de Chimie Bioorganique et des Syste` mes Mole´ culaires Vectoriels, Faculte´ des Sciences, 33 rue Louis Pasteur, F-84000 AVignon, France ReceiVed NoVember 6, 2006. In Final Form: January 19, 2007 The possibility of organizing detergent-solubilized membrane proteins in a plane within the core of Newton black films (NBFs) formed from fluorinated surfactants has been investigated. Fluorinated surfactants have the interesting characteristics of being poorly miscible with detergents and highly surface-active. As a result, when a membrane proteinsthe transmembrane domain of OmpA (tOmpA)ssolubilized by the nonionic detergent C8E4 (tetraethylene glycol monooctyl ether) was injected under a monolayer of fluorinated surfactant, C8E4 and tOmpA/C8E4 complexes remained confined to the subphase. Vertical, macroscopic NBFs were drawn, and their structure was investigated by means of X-ray reflectivity. Depending on experimental conditions, the protein was shown to organize into either one or two monolayers stabilized by two monolayers of fluorinated surfactant. Two different mechanisms of protein insertion were investigated: (i) attachment of polyhistidine-tagged tOmpA/C8E4 complexes to nickel-bearing polar groups born by a fluorinated surfactant and (ii) spontaneous diffusion into the surfactant films. Possible applications are discussed.
Introduction The aim of the present study was to explore the possibility of enclosing detergent-solubilized membrane proteins within thin, planar liquid films formed between two surfactant monolayers interacting with each other via long-range (DLVO) and/or shortrange (steric) forces. In free-standing liquid films, surfactant molecules adopt an organization opposite to that of lipids in biological membranes, with hydrophobic tails being exposed to the air phase and polar groups sheltered in the film interior. The type and the strength of the interactions depend on the distance between surfactant monolayers (i.e., the thickness of the film). Free-standing black films are of two types: common black films (CBFs) and Newton black films (NBFs).1 CBFs contain a liquid core between two surfactant monolayers. NBFs, the thinnest films, are completely drained and do not contain any liquid core. In previous studies,2,3 we established that the insertion of soluble proteins within the core of NBFs stabilized by nonionic surfactants or phospholipids can be achieved using different combinations of surfactants and proteins and by different mechanisms. We showed, in particular, that the protein can be integrated into the film either (a) via a process of diffusion from the bulk subphase or (b) after attachment of the protein to a surfactant monolayer at the air/water interface by either electrostatic or specific interactions. The choice of the surfactant can influence the * Corresponding author. E-mail:
[email protected]. † CEA-Saclay. ‡ CNRS & Universite ´ Paris 7. § Present address: Yale University, Department of Molecular Biophysics and Biochemistry, 266 Whitney Avenue, New-Haven, Connecticut 06520. | Universite ´ d’Avignon. (1) Exerowa, D.; Kruglyakov, P. M. Foam and Foam Films: Theory, Experiment, Application; Studies in Interface Science; Elsevier: Amsterdam, 1998; p 5. (2) Benattar, J.-J.; Nedyalkov, M.; Prost, J.; Tiss, A.; Verger, R.; Guilbert, C. Phys. ReV. Lett. 1999, 5297-5300. (3) Petkova, V.; Benattar, J.-J.; Nedyalkov, M. Biophys. J. 2002, 82, 541548.
mechanism of protein insertion. The final structures are NBFs composed of two monolayers of surfactant separated by a monolayer of protein. The present study aimed at investigating whether this process could be applied to highly hydrophobic proteins such as membrane proteins. Because NBFs are of nanometer thickness and extend over macroscopic (centimeter) sizes, they are amenable to studies using a variety of physical techniques, including X-ray reflectivity measurements. This opens the way to original investigations of the proteins that they enclose. Furthermore, the geometry thus achieved may provide a favorable starting point for 2D organization of the protein in the plane of the film. NBFs can be viewed as liquid-crystalline structures. The molecules comprising them are highly organized, and the forces in the lateral as well as the normal direction with respect to the film plane include strong steric forces. For this reason, it is not unreasonable to expect that membrane proteins confined within NBFs may exhibit a tendency to form 2D crystals. For the present experiments, NBFs were formed from fluorinated surfactants. Fluorinated surfactants (FSs) present an interesting combination of properties for such an application: (i) Fluorinated molecules are poorly miscible with hydrogenated ones.4-8 This property has many interesting consequences. For instance, (a) hydrogenated and fluorinated surfactants, when mixed in aqueous solution, can form separate micelles,7-9 and (b) biological lipids exhibit a low solubility in FSs.10-13 FSs, (4) Funasaki, N.; Hada, S. J. Phys. Chem. 1980, 84, 736-744. (5) Mukerjee, P. Colloids Surf., A 1994, 84, 1-10. (6) Lasic, D. Liposomes: From Physics to Applications; Elsevier Science B.V.: Amsterdam, 1993. (7) Kadi, M.; Hansson, P.; Almgren, M. Langmuir 2002, 18, 9243-9249. (8) Nakano, T-Y.; Sugihara, G.; Nakashima, T.; Yu, S.-C. Langmuir 2002, 18, 8777-8785. (9) Barthe´le´my, P.; Tomao, V.; Selb, J.; Chaudier, Y.; Pucci, B. Langmuir 2002, 18, 2557-2563. (10) Elbert, R.; Folda, T.; Ringsdorf, H. J. Am. Chem. Soc. 1984, 106, 76877692.
10.1021/la063249o CCC: $37.00 © 2007 American Chemical Society Published on Web 03/17/2007
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Figure 1. (A) Schematic model and estimated molecular dimensions of the tOmpA/C8E4 complex; (B) 3D structure of tOmpA25 (after PDB 1BXW); (C) chemical structures of the two fluorinated surfactants used in the present study, nonionic C6F13SOTHAM and nickel-bearing C8F17NTANi.
therefore, do not solubilize fats (i.e., they are not detergents);14-16 therefore, they are not cytolytic.15,17 Conversely, fluorinated dyes are insoluble in micelles of hydrogenated surfactants.18 This immiscibility has been exploited to form 2D crystals of a detergent-solubilized membrane protein adsorbed, by metal chelation, under a monolayer of derivatized fluorinated lipids spread at the air/water interface.19 (ii) Because they do not disrupt hydrocarbon/hydrocarbon interactions, FSs are better tolerated by membrane proteins than the hydrogenated surfactantssdetergentsstraditionally used in membrane biochemistry.15,20-21 (iii) The high surface activity of fluorinated surfactants22 and their limited miscibility with hydrogenated ones prevent the latter from effectively competing with FSs for adsorption at the air/ water interface.10,11 The present article addresses the establishment of conditions for membrane protein insertion into NBFs made from fluorinated surfactants. Free-standing, vertical, macroscopic NBFs were drawn from aqueous solutions comprising one (or two) FS(s), a membrane proteinstOmpA, the transmembrane region of outer membrane protein A from Escherichia coli,23-27sand the nonionic detergent C8E4 (tetraethyleneglycol monooctyl ether), which kept tOmpA water-soluble. X-ray reflectivity measurements show that the films are stabilized by two monolayers of FS, between which tOmpA organizes into one or two protein monolayers, depending on experimental conditions. Two different mechanisms of insertion were investigated, namely, (i) specific attachment of polyhistidine-tagged tOmpA/C8E4 complexes to nickel ions chelated by the polar moiety of a specially designed FS and (ii) spontaneous diffusion of the protein into the films. Materials and Methods Expression, Refolding, and Purification of tOmpA. The plasmid containing the coding sequence of the transmembrane domain (tOmpA, 171 residues) of outer membrane protein A from E. coli was kindly provided by G. E. Schulz (Freiburg University, Freiburgim-Brisgau, Germany). The protein contains three mutations (F23L, Q34K, and K107Y).23 An N-terminal tag comprising eight histidine residues was added to it.24 The construct (M ) 20 335 g‚mol-1) was overexpressed as inclusion bodies in E. coli. Purification and refolding procedures24,27 were similar to those previously described.25 The X-ray structure of tOmpA has been determined at 1.65 Å resolution.25-26 The domain is folded into an eight-stranded β-barrel with a diameter of 26 Å and a length of 57 Å (Figure 1A). The β-barrel comprises 102 residues. All strands are tilted at ∼45° relative
to the membrane normal. The extramembrane regions are comprised of long, mobile extracellular loops and short periplasmic turns (Figure 1B). In the present experiments, tOmpA was used as a complex with the nonionic detergent tetraethyleneglycol monooctyl ether (C8E4; M ) 306.4 g‚mol-1, critical micelle concentration (cmc) ) 8.5 mM). By analogy with other systems (see, for example, refs 27-29), C8E4 is expected to form a monolayer covering the transmembrane surface of tOmpA (Figure 1B), in keeping with the position of the six molecules of C8E4 revealed by X-ray crystallography.26 tOmpA/ C8E4 complexes have been studied in detail by size exclusion chromatography (SEC), analytical ultracentrifugation (AUC), and small-angle neutron scattering.24 The complex comprises one monomer of tOmpA and 86 ( 12 molecules of C8E4 for an overall mass of M ) 46 700 ( 3700 g‚mol-1. Its Stokes radius (RS), determined by SEC and AUC, is ∼35 Å (ref 24). Its translational diffusion coefficient Dt at 20-23 °C in aqueous buffers has been estimated to be 5 × 10-11 m2‚s-1 (by solution NMR)30 or (5.7-6.4) × 10-11 m2‚s-1 (by AUC and SEC).24 (11) Shibata, O.; Uamamoto, S. K.; Lee, S.; Sugihara, G. J. Colloid Interface Sci. 1996, 184, 201-208. (12) Krafft, M-P.; Guilieri, F.; Fontaine, P.; Goldmann, M. Langmuir 2001, 17, 6577-6584. (13) Funasaki, N. In Mixed Surfactant Systems; Ogino, K., Abe, M.. Eds.; Surfactant Science Series; Marcel Dekker: New York, 1993; Vol. 46, Chapter 5, p 145. (14) Pucci, B.; Maurizis, J-C.; Pavia, A.-A. Eur. Polym. J. 1991, 10, 11011106. (15) Chabaud, E.; Barthe´le´my, P.; Mora, N.; Popot, J.-L.; Pucci, B. Biochimie 1998, 80, 515-530. (16) Palchevsky, S. S.; Posokhov, Y. O.; Olivier, B.; Popot, J.-L.; Pucci, B.; Ladokhin, A. S. Biochemistry 2006, 45, 2629-2635. (17) Zarif, L.; Riess, J.-G.; Pucci, B.; Pavia, A.-A. Biomater,. Art. Cells, Immobilization Biotechnol. 1993, 21, 597-608. (18) Sta¨hler, K.; Selb, P.; Barthe´le´my, P.; Pucci, B.; Candau, F. Langmuir 1998, 14, 4765-4775. (19) Lebeau, L.; Lach, F.; Venien-Bryan, C.; Renault, A.; Dietrich, J.; Jahn, T.; Palmgren, M. G.; Ku¨hlbrandt, W.; Mioskowski, C. J. Mol. Biol. 2001, 308, 639-647. (20) Breyton, C.; Chabaud, E.; Chaudier, Y.; Pucci, B.; Popot, J.-L. FEBS Lett. 2004, 564, 312-318. (21) Lebaupain, F.; Salvay, A. G.; Olivier, B.; Durand, G.; Fabiano, A.-S.; Michel, N.; Popot, J.-L.; Ebel, C.; Breyton, C.; Pucci, B. Langmuir 2006, 22, 8881-8890. (22) Kissa, E. Fluorinated Surfactants: Synthesis, Properties, Applications; Surfactants Science Series; Marcel Dekker: New York, 1994; Vol. 50, Chapter 7, pp 264-282. (23) Pautsch, A.; Vogt, J.; Model, K.; Siebold, C.; Schulz, G. E. Proteins: Struct., Funct., Genet. 1999, 34, 167-172. (24) Zoonens, M. Doctorat d’Universite´, 2004, Paris-6. (25) Pautsch, A.; Schulz, G. E. Nat. Struct. Biol. 1998, 5, 1013-1017. (26) Pautsch, A.; Schulz, G. E. J. Mol. Biol. 2000, 298, 273-282. (27) Zoonens, M.; Catoire, L. J.; Giusti, F.; Popot, J.-L. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 8893-8898. (28) le Maire, M.; Champeil, P.; Møller, J. V. Biochim. Biophys. Acta 2000, 1508, 86-111.
Membrane Proteins in Newton Black Films
Langmuir, Vol. 23, No. 8, 2007 4305 Scheme 1. Synthetic Pathway of C8F17NTANi
Solutions of C8E4 and tOmpA were prepared in 20 mM Tris/HCl buffer at pH 8.0. Several concentrations of tOmpA were used in our studies (0.025, 0.05, 0.125, and 0.25 g‚L-1). The concentration of C8E4 was kept constant and equal to 6 g‚L-1 (i.e., about 2.5-fold the cmc). Analytical Procedures for Chemical Syntheses. The progress of the reactions and the homogeneity of the compounds were monitored by thin-layer chromatography (TLC, Merck 254 plates). Compounds were detected by exposure to UV light (254 nm) or by spraying a 5% sulfuric acid solution in methanol and/or a 5% ninhydrin solution in ethanol (detection of amine-containing compounds) and heating to 150 °C. Purification was performed by flash column chromatography over silica gel (Merck 60). All solvents were removed under vacuum. 1H, 13C, and 19F NMR spectra were recorded using a Brucker AC 250 instrument. 1H and 13C chemical shifts are given in parts per million relative to tetramethylsilane. Mass spectra were recorded on a DX 300 Jeol apparatus. All commercial solvents were distilled and dried according to standard procedures.
Organic Synthesis. C6F13SOTHAM (C6F13CH2CH2SOCH2CH2CONHC(CH2OH)3, Figure 1C) was synthesized as previously reported.31 C8F17NTANi (C8F17CH2CH2CONH(CH2)4CH(CO2-) N(CH2CO2-)2Ni2+, Na+, 2H2O; Figure 1C), was synthesized starting from N,N-dicarboxymethyl-N-benzyloxycarbonyl lysine (I), prepared as described by Hochuli et al.32 (Scheme 1). The three carboxylic functions were first protected as methyl esters using thionyl chloride in boiling methanol (II). After hydrogenolysis of the benzyloxycarbonyl group, the resulting amine (III) was reacted with a freshly prepared pentafluorophenyl ester of 2H,2H,3H,3H-perfluoroundecylic acid (IV), leading in good yield to the fluoroalkyl trimethyl ester. The ester functions were then hydrolyzed using a catalytic amount of sodium methylate in methanol. After neutralization, C8F17NTA (V) was isolated as a hygroscopic white powder in 56% overall yield. 1H NMR (250 MHz, CD3OD): δ 3.66 (s, 2H); 3.64 (s, 2H); 3.48 (t, 1H); 3.24 (m, 2H); 2.54 (m, 4H); 1.84-1.18 (m, 6H). 19F NMR (CD3OD): δ -82.4 (3F); -115.8 (2F); -122.9 (6F); -123.8 (2F); -124.5 (2F); -127.3 (2F). 13C NMR (CD3OD): δ 174.6;
(29) Ferna`ndez, C.; Hilty, C.; Wider, G.; Wu¨thrich, K. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 13533-13537. (30) Ferrage, F.; Zoonens, M.; Warschawski, D. E.; Popot, J.-L.; Bodenhausen, G. J. Am. Chem. Soc. 2003, 125, 2541-2545.
(31) Barthe´le´my, P.; Maurizis, J.-C.; Lacombe, J.-M.; Pucci, B. Bioorg. Med. Chem. Lett. 1998, 8, 1559-1562. (32) Hochuli, E.; Dobeli, H.; Schacher, A. J. Chromatogr. 1987, 411, 177184.
4306 Langmuir, Vol. 23, No. 8, 2007 174.5; 171.3; 65.2; 53.9; 38.9; 33.4; 29.3; 28.4; 24.7; 23.3. MS: [M (Na + H)]+ ) 759, [M (Na + NH4)]+ ) 775, [M (2Na + H)]+ ) 799, [M (3Na + H)]+ ) 803). Ni2+ complexation was achieved in good yield (83%) by the method described by Ve´nien-Bryan et al.33 Surface Tension Measurements. All solutions were prepared in water purified using a Milli-Q system (Millipore; resistivity ) 18.2 MΩ‚cm; surface tension ) 72.8 mN‚m-1). Measurements were carried out at 25 °C in 10 mM Tris/HCl buffer at pH 8. The surface tension was measured with a Wilhelmy plate using a Kru¨ss K12ST tensiometer controlled by Labdesk software (Kru¨ss, Germany). Under these conditions, the cmc of C6F13SOTHAM is 0.40 ( 0.05 mM, and its limiting surface tension (γcmc) is 16.0 ( 0.5 mN‚m-1; for C8F17NTA, cmc ) 0.025 ( 0.005 mM, and γcmc ) 20.5 ( 0.5 mN‚m-1. X-ray Reflectivity Measurements. X-ray reflectivity provides accurate information about the thickness and internal structure of vertical macroscopic (35 × 5 mm2) black films. Films drawn using a rectangular steel frame were irradiated with X-rays. After its formation, the film starts to drain, in a first step under gravity and, in a second step, because of surface forces acting between the two monolayers. Thinning of the film can be monitored by its color changes in visible light. Because the thickness of a black film is nanometric, visible light reflected on its two faces interferes destructively. X-rays, on the contrary, can undergo constructive interference. Reflectivity experiments were carried out using a sealed source (Cu KR1 line, λ ) 1.5405 Å) and a high-resolution diffractometer (Optix-Nonius), which have been described in detail elsewhere.34,35 They measure the ratio R(θ) ) I(θ)/I0, where I0 is the intensity of the incident beam and I(θ) is that of the reflected one, as a function of the incidence angle (or scattering wave vector Qz ) π sin θ/λ). Because of the high electron density gradient at air/film interfaces, reflectivity curves present well-defined interference fringes. The reflectivity profile gives access to the electron density profile along the film normal (z). Extracting information about the structure of the film relies on building a simplified model composed of parallel slabs with a uniform electron density. For each slab, the thickness, density, and interfacial roughness can be derived from the experimental profile thanks to an optical formalism that takes into account multiple reflections.36 In addition to the overall film thickness, the analysis also yields the area per surfactant molecule. The main advantage of free-standing films comes from their high electron density gradient at air/film interfaces, which generates reflectivity curves with well-defined interference fringes.
Results Fluorinated NBFs were drawn from monolayers formed at the air-water interface using either C6F13SOTHAM alone or a 2:1 mol/mol mixture of C6F13SOTHAM and C8F17NTANi. The latter FS carries a Ni2+ ion ligated by the NTA group (Figure 1C), onto which tOmpA can adsorb with high affinity via the polyhistidine tag that has been fused to its N terminus. We first describe the properties of NBFs formed in the absence of tOmpA and then compare them to those observed when tOmpA was present in the subphase. Protein-Free Films. The ability of the nickel-bearing surfactant to incorporate into NBFs was first examined by studying films formed from solutions containing either one or several of the surfactants C6F13SOTHAM (0.5 g‚L-1), C8F17NTANi (0.35 g‚L-1), and C8E4 (6 g‚L-1, i.e. about twice the cmc) at the respective concentrations to be used later in the presence of the protein. Experimental curves for the films drawn from C6F13(33) Venien-Bryan, C.; Balavoine, F.; Toussaint, B.; Mioskowski, C.; Hewat, E. A.; Helme, B.; Vignais, P. M. J. Mol. Biol. 1997, 274, 687-692. (34) Belorgey, O.; Benattar, J.-J. Phys. ReV. Lett. 1991, 66, 313-316. (35) Cuvillier, N.; Millet, F.; Petkova, V.; Nedyalkov, M.; Benattar, J.-J. Langmuir 2000, 16, 5029-5035. (36) Born, F.; Wolf, E. Principles of Optics, 6th ed.; Pergamon: London, 1984.
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Figure 2. X-ray reflectivity profiles of NBFs stabilized by C6F13SOTHAM (0.5 g‚L), with (9) or without (b) C8F17NTANi (0.35 g‚L), in the presence of C8E4 (6 g‚L-1). Solid lines are theoretical curves (fits) with the parameters given in Table 1. Qz is the scattering wave vector. Reflectivity measurements were performed after the films had reached their equilibrium thickness. Table 1. Thickness and Internal Structure of NBFs Drawn from Solutions Containing C6F13SOTHAM, C8F17NTANi, and/or C8E4 film parametersa sample composition
h (Å)
htails (Å)
hheads (Å)
hcore (Å)
C6F13SOTHAM C6F13SOTHAM+ C8E4 + Trisb C6F13SOTHAM + C8F17NTANi + C8E4 + Trisb C8F17NTANi + NaCl
41.6 41.0
12.1 11.8
7.0 6.9
3.4 3.6
68.1
11.8
10
24.5
51
11.3
9.9
8.5
a Parameters obtained by fitting reflectivity curves (cf. Figure 2): the five-layer film model comprised two layers for the fluorinated tails, two layers for the polar groups, and one layer for the aqueous core of the film. The total film thickness is thus h ) 2htails ( 2(hheads + hcore). b Data derived from the experiments shown in Figure 2. The concentrations of C6F13SOTHAM, C8F17NTANi, and C8E4 were 0.5, 0.35, and 6 g‚L-1, respectively.
SOTHAM solutions with and without C8F17NTANi are compared in Figure 2. Addition of the nickel-bearing surfactant caused a significant modification of the reflectivity curve, which exhibited more interference fringes and was shifted toward small incident angles as compared to those for films composed of pure C6F13SOTHAM. These changes indicate film thickening and suggest structural modifications. Theoretical curves were fitted to experimental ones using a five-layer-film model:35 two layers for the hydrophobic moieties (index “tails”), two for the polar groups (“heads”), and one (“core”) for the interior of the film. The overall film thickness is thus h ) 2 × htails ( 2 × hheads + hcore. For films of pure surfactants, we assumed the composition of the central layer to be identical to that of the subphase, and its electron density was kept equal to that of water. The other parameters were varied according to different possible molecular arrangements and the corresponding changes in the area per molecule. Layer thicknesses derived from the analysis are listed in Table 1, as well as those obtained for films drawn from onecomponent solutions of either C6F13SOTHAM or C8F17NTANi. Note that the net negative charge of the NTANi polar head required the addition of an electrolyte (NaCl) to stabilize the film drawn from the one-component C8F17NTANi solution so as to shield the electrostatic repulsion between polar head layers. A comparison of the properties of the NBFs drawn under these various
Membrane Proteins in Newton Black Films
Langmuir, Vol. 23, No. 8, 2007 4307 Table 2. Thickness and Internal Structure of NBFs Stabilized by C6F13SOTHAM, C8F17NTANi, and C8E4 in the Presence of tOmpA (0.025 g‚L-1) film parametersa incubation time (h)b
h (Å)
htails (Å)
hheads (Å)
hcore (Å)
2 24
71 105
11.4 11.4
9.7 7.9
28.8 66.6
a Parameters derived by fitting the reflectivity curves shown in Figure 3. b Films were drawn either 2 h or 24 h after forming the air/water interface.
Figure 3. X-ray reflectivity profiles of films made from a solution containing C6F13SOTHAM, C8F17NTANi, C8E4, and tOmpA (0.025 g‚L-1), drawn either 2 h (9) or 24 h (2) after creating the air/ solution interface. Solid lines are theoretical curves (fits) with the parameters shown in Table 2. The reflectivity curve for a film stabilized by the surfactants alone is added for comparison (b).
conditions allows one to sort out the influence of each surfactant on film properties. As found before,37 the film formed from pure C6F13SOTHAM is a very thin NBF with an ∼3.5-Å-thick internal layer, consistent with the presence of water of hydration around the polar groups of the surfactant. Films drawn from mixed C6F13SOTHAM + C8E4 solutions were indistinguishable from those drawn using pure C6F13SOTHAM (Table 1). This is consistent with the expected exclusion of C8E4 from the air/water interface and furthermore indicates that C8E4 micelles (RH ) 2.5 nm, aggregation number ∼80)38 are expelled from the film during drainage. The increased thickness hheads observed in the presence of C8F17NTANi is consistent with the addition of the NTA/Ni group to the polar moiety of the fluorinated surfactant (Figure 1C). However, the most striking difference between the films is the thickness of their central layer. The much larger hcore in the film drawn from the solution containing C6F13SOTHAM + C8F17NTANi + C8E4 (in Tris buffer) must result from the existence of a significant electrostatic contribution to the disjoining pressure acting between polar heads (repulsive electrostatic interactions). This effect indicates that the films formed from mixed C6F13SOTHAM + C8F17NTANi solutions do comprise the nickelbearing surfactant, a prerequisite to incorporating histidine-tagged proteins into their core. Nevertheless, the thickening induced by the presence of the NTANi group (∼21 Å) remains much less than the size of the tOmpA/C8E4 complex (RS ≈ 35 Å), making it straightforward to detect any incorporation of the complex. Membrane Protein Insertion Driven by Metal Chelation. In this experimental configuration, tOmpA/C8E4 complexes are first allowed to adsorb from the subphase under a mixed monolayer containing both C6F13SOTHAM and C8F17NTANi. Two monolayers are then brought together to form the NBF, trapping the protein between them. In an earlier study involving the adsorption of a histidine-tagged membrane protein under a monolayer of nickel-bearing lipids, it was noted that successful 2D crystallization of the protein required an incubation period of 24 h.19 In the present experiments, films were drawn from the monolayer either 2 or 24 h after the formation of the air/solution interface. The corresponding reflectivity curves are shown in Figure 3. In the presence of tOmpA, films drained very slowly, and their thickness reached equilibrium only after 2 to 3 h. The experimental curves and thickness values given in Figure 3 and (37) Barthe´le´my, P.; Cuvillier, N.; Chaudier, Y.; Benattar, J.-J.; Pucci, B. J. Fluorine Chem. 2000, 105, 95-102. (38) Corti, M.; Minero, C.; Degiorgo, V. J. Phys. Chem. 1984, 88, 309-317.
Figure 4. Comparison of X-ray reflectivity profiles of films made from solutions of C6F13SOTHAM, C8F17NTANi, and C8E4 in the presence of tOmpA at either 0.025 (2) or 0.25 g‚L-1 ([). Solid lines are theoretical curves (fits) with the parameters shown in Table 3. Table 3. Thickness and Internal Structure of NBFs Stabilized by C6F13SOTHAM, C8F17NTANi, and C8E4 in the Presence of tOmpA at Different Concentrations film parameters solutions
h (Å)
htails (Å)
hheads (Å)
hcore (Å)
no tOmpA + tOmpA (0.025 g‚L-1)a + tOmpA (0.05 g‚L-1) + tOmpA (0.125 g‚L-1) + tOmpA (0.25 g‚L-1)a
68.1 105 110 102 157
11.8 11.4 11.4 11.5 10.6
10 7.9 10 9.7 9
24.5 66.6 67 59.4 118
a
Parameters derived by fitting the reflectivity curves shown in Figure 4.
Table 2 correspond to equilibrium films. Even when drawn only 2 h after forming the monolayer, NBFs were thicker than those formed in the absence of protein. Leaving tOmpA to adsorb for 24 h before drawing the films resulted in still thicker NBFs, obviously incorporating more protein, whatever the protein concentration used. (See below.) The molar ratio tOmpA/C8F17NTANi (R) was modulated by changing the protein concentration from 0.025 g‚L-1 (R ) 1:330) to 0.25 g‚L-1 (R ) 1:33) while keeping the concentration of the surfactants constant (Figure 4). Increasing the bulk protein concentration from 0.025 to 0.125 g‚L-1 had no significant effect on the film properties, with the equilibrium film thickness having about the same value (∼100 Å) at all tOmpA concentrations (Table 3). To fit the data, we assumed that the distribution of tOmpA/C8E4 complexes was restricted to the film core, and we varied both the thickness and electron density of the central film layer. The thickness of the layers comprising the hydrophobic chains of the fluorinated surfactants did not change with the protein concentration. Thus, the presence of the protein affects neither the composition of the monolayers nor their molecular arrangement. Up to R ) 1:66, the thickness of the core remains remarkably constant. At 60-67 Å, it is close to twice the Stokes
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Figure 5. Evolution over time of the X-ray reflectivity profile of a film made from solutions of C6F13SOTHAM and C8E4 in the presence of 0.25 g‚L-1 tOmpA. Solid lines are theoretical curves (fits) with the parameters shown in Table 4. The different reflectivity curves describe the evolution of one and the same film 30 min ([), 3 h (9), and 17 h (2) after the film had turned entirely black. The reflectivity curve for a film stabilized by the surfactants only is added for comparison (b). Table 4. Thickness and Internal Structure of NBFs Stabilized by C6F13SOTHAM and C8E4 in the Presence of tOmpA (0.25 g‚L-1) at Different Times after the Film Was Formed film parametersa incubation time
h (Å)
htails (Å)
hheads (Å)
hcore (Å)
no tOmpA t ) 30 min t ) 180 min t ) 17 h
41.0 57 48 91
11.8 11.3 11.2 12
6.9 8.5 8.6 7.3
3.6 17.5 8.4 52.6
a Parameters derived by fitting the reflectivity curves shown in Figure 5.
radius of the tOmpA/C8E4 complex (∼35 Å).24 At 0.25 g‚L-1 tOmpA in the subphase (R ) 1:33), the thickness of the film core increased up to ∼120 Å (i.e., about twice the hydrodynamic diameter of the tOmpA/C8E4 complex). Under these conditions, however, the stability of the film was drastically reduced (to less than 3 h), and its thickness never stabilized. It may well be, therefore, that drainage was not complete by the time of the measurements. Protein Insertion by Free Diffusion. For these experiments, we built upon our previous experience with soluble proteins2,3 and resorted to NBFs of pure C6F13SOTHAM. As shown before, such films have a high stability (they are stable over 2 days), and they are very thin (40-45 Å)38 (Table 1). Experimental results for a film drawn from a mixed solution of C8E4 (6 g‚L-1), C6F13SOTHAM (0.5 g‚L-1), and tOmpA/C8E4 complexes (0.25 g.L-1 tOmpA) in Tris/HCl buffer are shown in Figure 5. The different reflectivity curves correspond to one and the same film scanned at different times after its formation and follow its evolution over time. The parameters derived thereof are listed in Table 4. An experimental curve for a film drawn from a solution without protein is shown on the same graph, and the corresponding parameters (from Table 1) are recalled in Table 4 for comparison. In the absence of protein, drainage was very fast, with the thickness of the film stabilizing after only 10 min. In the presence of tOmpA/C8E4 complexes, the rate of drainage decreased considerably, with the film reaching its minimal thickness 3 h after its formation. Drainage can be observed in Figure 5 as a reduction of the number of interference fringes and their shift to higher incident angles from t ) 30-180 min after the film appeared to be entirely black. The slow drainage is obviously
related to an increase in solution viscosity in the core of the film, which is due to the presence of protein/detergent complexes. After being drained, the film thickened again over the following 17 h. This can be observed in Figure 5 as a modification of the reflectivity curves and a shift of the interference fringes back toward small incident angles. The film parameters show a significant increase in the final film thickness (Table 4). The difference between the final thickness of the films made from solutions with and without protein is ∼50 Å and is mainly due to a thickening of the core. The increase again is close to although slightly less than the hydrodynamic dimensions of the tOmpA/ C8E4 complex.24 (Note that hcore is a mean value, which averages thickness variations.) The expected time for protein diffusion was estimated using the diffusion coefficient value Dt ) 5 × 10-11 m2‚s-1 for the tOmpA/C8E4 complex determined by NMR30 and a film height of 3 mm. The calculated value of 19-22 h is in good agreement with the observed swelling time of the film (17 h). Thus, it seems reasonable to conclude that it is the diffusion of tOmpA/C8E4 complexes into the film that is responsible for its thickening and that the final structure is an NBF with a protein monolayer in its core. In contrast to nickel-mediated protein insertion, it is difficult here to predict the protein orientation within the film because there is no specific protein/fluorinated surfactant interaction. One can, however, expect the formation of hydrogen bonds between the hydrophilic extramembrane residues of tOmpA and the polar hydroxyl groups of the fluorinated surfactant C6F13SOTHAM. Thickening was observed only in the presence of tOmpA and was perfectly reproducible with different films drawn from the same solution as well as with films drawn from different solutions under similar conditions. However, films made from fresh solutions were very thin (same thickness as films of pure surfactants), and it was obvious that they did not contain any protein. Two months later, films drawn from the same solutions were as thick as those obtained from fresh solutions in the presence of C8F17NTANi. Films made, in the absence of protein, from solutions of C6F13SOTHAM + C8E4 in Tris buffer had the same thickness whether drawn immediately after the preparation of the solutions or 2 months later. Thus, it is highly likely that the changes observed with storage time are related to the aging of the protein. The alteration (possibly by proteolysis) that induces the migration of the aged protein into the films was not investigated.
Discussion X-ray reflectivity measurements unambiguously indicate that one (or two) monolayers(s) of tOmpA can be inserted between the two monolayers of fluorinated surfactant that stabilize the NBF. Trapping of tOmpA/C8E4 complexes was achieved by two different mechanisms: (a) by specific interactions, after the histidine-tagged membrane protein had been bound by metal chelation to a nickel-bearing FS incorporated into the monolayer from which the films are drawn, and (b) by spontaneous diffusion of the complexes into preformed NBFs, in the absence of specific adsorption. The amount of protein/detergent complexes captured in the preformed films using metal chelation was found to depend on the molar ratio R of tOmpA to nickel-bearing surfactant: a single protein layer was formed at low protein concentrations (R ) 1:330 to 1:66) and a double layer was formed at high concentration (R ) 1:33). The time allowed for surface saturation before the film is drawn is another important factor: films contain more protein after a long incubation period (a day). The probable structure of the films is schematically described in Figure 6. Before the film is formed, the air/water interface is occupied by
Membrane Proteins in Newton Black Films
Figure 6. Schematic representation of the arrangement of tOmpA with respect to the surfactants (A) under monolayers at the air/water interface, (B) in an NBF formed at low protein concentration (0.025 g‚L-1), and (C) in an NBF formed at high protein concentration (0.25 g‚L-1).
the monolayer of C6F13SOTHAM and C8F17NTANi at a molar ratio (if similar to that in the subphase) of ∼2:1. Under the mixed surfactant monolayer, tOmpA/C8E4 complexes are expected to bind by metal chelation to the Ni2+ ions carried by C8F17NTANi (Figure 6A). X-ray reflectivity measurements show that after the formation of the NBF membrane proteins are present in its core. The final thickness of the film core at [tOmpA] ) 0.025-0.125 g‚L-1, 60-67 Å, is close to the hydrodynamic diameter of one tOmpA/C8E4 complex (∼70 Å) or to the length of a protein molecule (∼57 Å). This suggests the presence of a single continuous membrane protein monolayer between the fluorinated film walls, either with an intercalated or random arrangement, or, perhaps, the formation of microdomains (Figure 6B). At higher protein concentration (0.25 g‚L-1), the final thickness of the film core reached 118 Å. This is about twice the length of tOmpA, suggesting the presence of two protein monolayers (Figure 6C). When they reached this thickness, however, the films ruptured. Membrane protein insertion can also proceed through a freediffusion process without the need for specific adsorption. Spontaneous insertion, however, was observed only after the protein/surfactant solutions had been stored for a couple of months. Control experiments indicate that this effect is due to the aging of tOmpA. The alteration that induces the migration of the aged protein into the films was not identified. The novel experimental configuration described here has many possible applications. For one thing, the process observed with
Langmuir, Vol. 23, No. 8, 2007 4309
membrane proteins could conceivably be extended to other types of detergent-stabilized hydrophobic particles (e.g., nanodots or nanotubes). With respect to membrane proteins, the formation of large planar arrangements, particularly when the adsorption is mediated by metal chelation and the proteins are likely to be at least partially oriented, opens up some interesting perspectives. It ought to be possible, for instance, to follow and to quantify, by X-ray reflectivity, the binding to the immobilized membrane proteins of macromolecular or electron-dense ligands. Provided they affect the thickness of the film core, it should be possible as well to detect and measure membrane protein conformational changes induced either by ligand binding or by other stimuli, such as pH, light, or other environmental changes. Because of the large surface of the films, it may also be possible to use them to follow spectroscopically processes such as diffusion within the monolayer of membrane proteins of ligands, protons, electrons, excitons, and so forth. A particularly attractive application would be to use NBFs as a matrix for growing 2D membrane protein crystals. Crystal formation ought to be facilitated by the planar arrangement imposed upon membrane proteins by the geometry of the experiment, the orientation with respect to the plane of the film induced by specific adsorption, molecular crowding, and the strong steric forces that result from it. Approaches other than X-ray reflectivity (e.g., electron microscopy after transfer of the NBF onto a grid) would, however, be needed to probe the films for in-plane organization. Acknowledgment. Particular thanks are due to C. Ve´nienBryan (Oxford University) and C. Breyton (UMR 7099) for their critical reading of a first version of the manuscript and to G. E. Schulz (Freiburg-im-Breisgau) for the gift of the tOmpA plasmid. M.Z. was the recipient of a doctoral fellowship from the Ministe`re de la Recherche et de la Technologie. This work was supported by the CNRS, Universite´ Paris-7, Universite´ d’Avignon et des Pays du Vaucluse, the Commissariat a` l’Energie Atomique, and EU STREP “Innovative Tools for Membrane Structural Proteomics”. LA063249O
