Unexpected Stability of Phospholipid Langmuir Monolayers Deposited

Langmuir 2007, 23, 12959-12965

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Unexpected Stability of Phospholipid Langmuir Monolayers Deposited on Triton X-100 Aqueous Solutions Philippe Fontaine,*,†,⊥ Marie Claude Faure´,‡,§ Franc¸ ois Muller,§,| Mathieu Poujade,§,∇ Jean-Se´bastien Micha,£ Franc¸ ois Rieutord,£ and Michel Goldmann‡ Synchrotron SOLEIL, L’Orme des Merisiers, Saint Aubin, BP48, 91192 Gif sur YVette Cedex, France, Institut des NanoSciences de Paris (INSP, UMR 7588 CNRS UniVersite´ Paris VI and VII), Campus Boucicaut, 140 Rue de Lourmel, 75015 Paris, France, UFR Biome´ dicale des Saint-Pe` res, UniVersite´ Rene´ Descartes, 45, Rue des Saints Pe` res, 75231 Paris Cedex 06, France, and CEA-Grenoble/DRFMC/SI3M, 17 rue des Martyrs, F-38054 Grenoble Cedex and ESRF, BP 220, F-38043 Grenoble Cedex, France ReceiVed May 10, 2007. In Final Form: August 30, 2007 We studied at the molecular level the interaction between neutral detergent Triton X-100 aqueous solution and a phospholipid Langmuir monolayer deposited on top using surface pressure measurement and grazing incidence X-ray diffraction (GIXD). Macroscopically, the detergent-phospholipid system follows the Gibbs law. However, GIXD shows that the detergent and the phospholipid segregate at the interface. The molecular organization of pure phospholipid domains is imposed by the detergent through surface pressure. Compression and expansion of the surface monolayer system in its final state reveal the stability of the phospholipids domains against dissolution by the detergent in the subphase, even above the detergent cmc. This resistance to dissolution is suppressed by an expansion of the monolayer.

1. Introduction The study of the interaction between soluble and insoluble surfactant exhibits several interests either for fundamental research or for applications in industrial processes. In physical chemistry, such systems were studied in the case of aqueous solutions of soluble surfactants with micelles or vesicles of insoluble molecules.1-4 At interfaces, the penetration of insoluble surfactant mono- or bilayers by soluble surfactant dissolved in the liquid phase has been studied in many configurations.5-14 Consequently, the behavior of such systems is also a very active field of theoretical physical chemistry.6-8 However, most of these experimental and theoretical studies deal with penetration of insoluble layers by neutral or charged surfactants below the cmc * To whom correspondence should be addressed. E-mail: [email protected]. † Synchrotron SOLEIL. ‡ Institut des NanoSciences de Paris. § Universite ´ Rene´ Descartes. £ CEA-Grenoble/DRFMC/SI3M and ESRF. ⊥ Also at INSP. | Present address: Physical Chemistry, University of Graz, Heinrichstrasse 28, A-8010 Graz, Austria. ∇ Present address: Laboratoire de Physico-Chimie “Curie” (UMR CNRS 168), Institut Curie, Section de Recherche, 11, Rue Pierre et Marie Curie, F-75231 Paris Cedex 05, France. (1) de la Maza, A.; Parra, J. L. Biophys. J. 1997, 72, 1668. (2) Drummond, C. J.; Warr, G. G.; Grieser, F.; Ninham, B. W.; Evans, D. F. J. Phys. Chem. 1985, 89, 2103. (3) de la Maza, A.; Parra, J. L. Biochem. J. 1997, 303, 907. (4) Kragh-Hansen, U.; Le Maire, M.; Møller, J. V. Biophys. J. 1998, 75, 2932. (5) Morandat, S.; El Kirat, K. Langmuir 2006, 22, 5786. (6) Sundaram, S.; Stebe, K. J. Langmuir 1996, 12, 2028.; 1997, 13, 1729. (7) Fainerman, V. B.; Makievski, A. V.; Vollhardt, D.; Siegel, S.; Miller, R. J. Phys. Chem. B 1999, 103, 330. (8) Fainermann, V. B.; Vollhardt, D.; Roth, A.; Frieke, M.; Dolkner, D. J. Phys. Chem. B 2004, 108, 16163. (9) Cabrerizo-Vilchez, M. A.; Wege, H. A.; Holgado Terriza, J. A. Neumann, A. W. ReV. Sci. Instrum. 1999, 70, 2438. (10) Pethica, B. A. Trans. Faraday Soc. 1955, 51, 1402. (11) Gaines G. L. Insoluble Monolayers at Liquid Gas Interfaces; J. Wiley and Sons: New York 1966. (12) Jiang, Q.; O’Lenick, J.; Valentini, J. E.; Chiew, Y. C. Langmuir 1995, 11, 1138. (13) Hu, B.; Mi, L. Z.; Sui, S. F. Thin Solid Films 1998, 327-329, 69. (14) Nyholm, T.; Slotte, J. P. Langmuir 2001, 17, 4724.

where the dissolution of the insoluble species in the subphase, mediated by the presence of supramolecular assemblies in the bulk solution, is not expected. Moreover, no surface microscopic measurements have been performed, leading to a restrained macroscopic description. The understanding of such a system also has many interests in biophysics and biological processes. Indeed, in a lot of situations, a soluble surfactant, also called a detergent, is in interaction with a membrane or a monolayer made of a second surfactant, usually an insoluble phospholipid. This is especially the case during the extraction process of membrane proteins from the cell membrane15 or during the two-dimensional crystallization of membrane proteins.16 In this last case, the extracted membrane proteins are embedded in detergent micelles to dissolve them in water and to avoid their denaturation, keeping an amphiphilic environment around. In order to apply to the case of insoluble membrane proteins, the efficient technique of twodimensional crystallization of soluble proteins below Langmuir monolayers17-22 (monomolecular layers of insoluble surfactant at a liquid-air interface), attempts were made to perform twodimensional crystallization below Langmuir monolayers deposited on top of the detergent protein solution. If this technique appears successful in a few cases,16 the lack of understanding of the detergent effects on Langmuir monolayer makes such technique as hardly predictable. In this paper, we present a first surface microscopic study of the penetration of phospholipid Langmuir monolayer by neutral detergent above and below the detergent cmc. We focus on the structural changes at the molecular level in the monolayer induced by the presence of the detergent in the subphase. Indeed, to go (15) Lambert, O.; Le´vy, D.; Ranck, J. L.; Leblanc, G.; Rigaud, J. L. Biophys. J. 1998, 74, 918. (16) Chami, M.; Pehau-Arnaudet, G.; Lambert, O.; Ranck, J.-L.; Le´vy, D.; Rigaud, J. L. J. Struct. Biol. 2001, 133, 64. (17) Kornberg, R. D.; Darst, S. A. Curr. Opin. Struct. Biol. 1991, 1, 642. (18) Glaeser, R. M. Ann. ReV. Phys. Chem. 1985, 36, 243. (19) Sowadski, J. M. Curr. Opin. Struct. Biol. 1994, 4, 761. (20) Jap, B. K.; Zulauf, M.; Scheybani, T.; Hefti, A.; Baumeister, W.; Aebi, U.; Engel, A. Ultramicroscopy 1992, 46, 45. (21) Fromherz, P. Nature 1971, 231, 267. (22) Uzgiris, E. E.; Kornberg, R. D. Nature 1983, 301, 125.

10.1021/la701293n CCC: $37.00 © 2007 American Chemical Society Published on Web 11/20/2007

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further in this system, three complementary measurements were performed: (1) the time evolution of the surface pressure (reduction of the surface tension due to the presence of surfactant at the interface) since detergent injection, (2) the surface pressure evolution, measured by compression and expansion of the monolayer (π-A isotherms), after reached the stationary state in the adsorption process, and (3) the molecular organization at the air-solution interface studied using grazing incidence X-ray diffraction (GIXD). 2. Experimental Section 2.1. Molecule and Sample Preparation. The studied molecule is DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine). DMPE contains two hydrocarbon chains of 16 carbons each and has been chosen for its polymorphism, which has been extensively investigated.23,24 The Langmuir monolayer of DMPE exhibits at room temperature a liquid expanded (LE) phase at low surface pressure (π) and a liquid condensed (LC), organized phase at higher π values. DMPE was purchased from Sigma with purity better than 99% and used as received. Spreading solutions of 1 mmol‚L-1 are made using chloroform/methanol (Fisher, certified HPLC) 9:1 mixtures. The detergent is a neutral surfactant: isooctylphenoxy-polyethoxyethanol, classically referred to by its trademark name Triton-X100. It contains an average of 9.5 oxyethylene units per molecule. Since its applications are numerous, this detergent has been extensively studied.14,25-27 Its cmc ranges between 0.1 and 0.2 mmol‚L-1. TritonX100 was purchased from Sigma (ref T9284) and used without further purification. It was dissolved in ultrapure water (resistivity >18 MΩ‚cm, Millipore MilliQ system) to form a native solution of concentration 150 mmol‚L-1. This ultrapure water was also used for the water subphase of the Langmuir monolayers. Above the cmc, the surface tension of a Triton-X100 solution is constant and measured at γX100 ) 29.8 ( 0.1 mN‚m-1 which corresponds to a surface pressure πX100 ) 43 ( 0.1 mN‚m-1 at 20 °C. This value is in agreement with previous results.13 2.2. Surface Pressure Measurement. Monolayers were prepared on a dedicated Langmuir trough. Actually, when detergent is added to the subphase of a Langmuir monolayer, the PTFE edges of the trough are wetted by the detergent solution, leading above the cmc to an overflowing of the trough. In order to overcome this difficulty, we use a homemade Langmuir trough with a PTFE ribbon as the barrier. Such a trough does not need to be completely filled with water, avoiding the possibility of overflowing. The volume of water in the trough was fixed at 1 L. Compressions and expansions were made at a rate of 0.026 nm2‚min-1. All experiments were performed at room temperature (20 °C). Surface tension was measured by the Wilhelmy plate method.11 The plate is made using filter paper 2 mm large and 0.1 mm thick. It hangs to a surface pressure sensor (Riegler and Kirstein Gmbh, Wiesbaden, Germany). The accuracy of the measurement was better than 0.1 mN‚m-1. In this work, surface pressure (π) is defined as the reduction of the surface tension (γ) of pure water (γ0) due to the presence of a surfactant (detergent or lipid) monolayer at the interface: π ) γ0 - γ. For simplicity and clarity, we will always refer in the following to surface pressure (π) instead of surface tension to describe the state of the solution-air interface. 2.3. Detergent Injection in the Water Subphase. The quicker and easier way to add the detergent in the water subphase would be to inject in the subphase a small volume (typically 100 µL) of the native detergent solution (150 mmol‚L-1). However, such a procedure (23) Albrecht, O.; Gruler, H.; Sackmann, E. J. Phys. (Paris) 1978, 39, 301; Brezesinski, G.; Dietrich, A.; Struth, B.; Bo¨hm, C.; Bouwman W. G.; Kjaer, K.; Mo¨hwald, H. Chem. Phys. Lipids 1995, 76, 145. (24) Helm, C. A.; Tippman-Krayer, P.; Mo¨hwald, H.; Als-Nielsen, J.; Kjaer, K. Biophys. J. 1998, 60, 1457. (25) Robson, R. J.; Dennis, E. A. J. Phys. Chem. 1977, 81, 1075. (26) Goyal, P. S.; Menon, S. V. G.; Dasannacharya, B. A.; Thiyagarajan, P. Phys. ReV. E 1995, 51, 2308. (27) Carnero Ruiz, C.; Molina-Bolivar, J. A.; Aguiar, J.; MacIsaac, G.; Moroze, S.; Palepu, R. Langmuir 2001, 17, 6831.

Fontaine et al. leads to an auto-oscillating surface tension due to a switching mechanism between diffusion and convection within the solution, as described by Kovalchuk and co-workers.28 To suppress such spurious oscillations, we exchange the water subphase (by using a peristaltic pump). The detergent is first dissolved into a reservoir at a concentration adjusted to reach the requested final concentration in the subphase (in the range 0.005-0.5 mmol‚L-1). The pump is connected to the reservoir and to the trough using clean silicone tubes. The exchange takes place over 15 min at a flow of 140 mL‚min-1. Such experimental procedure suppresses most of the oscillations. 2.4. GIXD. Below the cmc, GIXD measurements were carried out at the D41B beamline of the DCI storage ring of the Laboratoire pour l’Utilisation du Rayonnement Electromagne´tique (LURE, Orsay, France). We use the usual Langmuir trough of the beamline for this measurement since no overflowing of the trough occurs. The X-ray beam was monochromatized to λ ) 0.1646 nm (7.55 keV) by the bent, monocrystalline surface of a Germanium plate (111), then collimated by a set of narrow horizontal and vertical slits and finally deflected downward by a glass mirror. The vertical incidence angle on the water surface was Ri ) 2.13 mrad, less than the critical angle for total external reflection (Rc ) 2.85 mrad for the air-water interface at the working energy). This glancing incidence method is now standard to minimize the amount of scattering by the subphase with respect to the surface signal.29,30 The acquisition time for a typical scan was 20 min. Above the cmc, due to the wetting of the trough edges by the detergent solution, we use the dedicated Langmuir trough with PTFE ribbon as barrier. In order to perform GIXD measurements, this trough is equipped with two Kapton windows, one for the incident beam and one for the diffracted beam. The trough is filled with water to cover these windows at half-height. The wetting properties of such windows lead to a climbing meniscus on the windows. Thus, the incoming and the diffracted X-ray beam have to cross the water meniscus to reach the interface and the detector, respectively. In order to reduce the impact of X-ray absorption by the water and maximize the signal-to-noise ratio, we performed GIXD using the high-energy, high-flux X-ray beam available at the CRG-IF (BM32) beamline at the ESRF. Actually, at 20 keV, the photons transmission of 1 mm of water is 93% compared to 36% at 8 keV.31 The white X-ray beam from the bending magnet was monochromatized to λ ) 0.062 nm (20 keV) by a double-crystal monochromator (Si 111) performing also horizontal focusing. Vertical focusing is provided by a bend mirror prior to the monochromator. The monochromatic beam is collimated by a set of narrow horizontal and vertical slits which defines the final horizontal and vertical size of the beam to, respectively, 1 mm and 30 µm. The incoming beam is finally deflected downward by a glass mirror. The small vertical size was chosen to minimize the X-ray footprint on the liquid subphase which is rather large considering the small incidence angle (θi ) 0.8 mrad) needed for such high-energy X-ray. The Langmuir trough was installed on the multitechnique goniometer (GMT) of the beamline. The typical acquisition time was less than 30 min. Despite this adapted “wetting liquid” setup, the GIXD experiment above the cmc remains difficult due to the wetting properties evolution of the liquid forming the subphase after the injection of the detergent. This wetting properties change could be attributed mainly to surface pressure evolution. Since the X-ray beams (incident and diffracted) have to cross the liquid meniscus, the signal-to-noise ratio is low. Indeed, the signal-to-noise ratio is estimated to be 3.4 below the cmc with the classical trough and 0.4 above the cmc with the “wetting liquid” setup. Moreover, since the X-ray scattering by the water (28) Kovalchuk, V. I.; Kamusewitz, H.; Vollhardt, D.; Kovalchuk, N. M. Phys. ReV. E 1999, 60, 2029. (29) Jacquemain, D.; Grayer-Wolf, S.; Leveiller, F.; Deutch, M.; Kjaer, K.; Als-Nielsen, J.; Lahav, M.; Leiserowitz, L. Angew. Chem., Int. Ed. Engl. 1992, 31, 130. (30) Fontaine, P.; Goldmann, M.; Bordessoules, M.; Jucha, A. ReV. Sci. Instrum. 2004, 75, 3097. (31) Data from Center of X-Ray Optics (CXRO) of the E. O. Lawrence Berkeley National Lab., http://www-cxro.lbl.gov/optical_constants/.

Unexpected Stability of Phospholipid Langmuir Monolayers meniscus is mainly out of plane, we are not able to measure the out-of-plane peak profile, as will be shown in the following. However, we succeeded with this adapted setup and high-energy photons to measure rod integrated in-plane diffraction peak above the cmc and deduce the chain organization from comparison with peaks positions on pure water or below the cmc. In both experiments, the air scattering was reduced by enclosing the Langmuir trough in a gastight box flushed with Helium gas. The intensity of the incident beam (I0) was monitored with an NaI detector measuring the scattered signal by a Kapton window crossing the incident beam, whereas the intensity of the diffracted beam (Ic) was measured with a vertical gas-filled (Xe-CO2) position-sensitive detector. The out-of-plane signal was measured at each in-plane wave vector (Qxy). A 1.4 mrad Soller slits collimator was positioned in front of the PSD window. Its acceptance corresponds to a scattering wave vector resolution of 0.07 nm-1 at Qxy ) 15 nm-1. In classical Langmuir monolayers like fatty acids or phospholipids, the main contribution to the scattered signal comes from the alkyl chains.29,32 All the diffraction peaks presented here have been subtracted from a linear background. Peak positions, full width at half maximum (fwhm), and intensity have been obtained by fitting the peaks by Lorentzian functions since diffraction peaks do not appear resolution limited.

3. Results For each experiment, we applied the following procedure: phospholipid monolayer deposited on pure water subphase is compressed up to the initial surface pressure π0. This defines the initial state of the monolayer. At time t ) 0, the pure water subphase is then exchanged with the Triton X-100 solution to reach the concentration cTX100. Then, we record the time evolution of surface pressure π(t) from the initial surface pressure π0 to the final surface pressure π∞. The film is then expanded and recompressed. Among all the performed experiments, we present here two typical behaviors of this system: below and above the Triton X-100 cmc. 3.1. Detergent Concentration below the cmc. Figure 1A shows the surface pressure behavior of DMPE monolayer, initially in the LC phase (π0 ) 12 mN‚m-1), and after the injection of Triton-X100 in the subphase at concentration c ) 0.005 mmol‚L-1. The full line at low surface pressure and high area per molecule is the compression of the monolayer up to π ) 12 mN‚m-1 on the water subphase. The subphase is then exchanged with the Triton-X100 solution. The evolution of surface pressure with time is depicted in the inset of Figure 1A. It shows an increase of surface pressure up to a plateau value π∞ ) 36 ( 0.5 mN‚m-1. This equilibrium pressure is reached in about 20 h despite spurious oscillations. Then, the monolayer is expanded to larger area per molecule. One observes that the surface pressure decreases with surface density (area per molecule). When the maximum area is reached, the pressure spontaneously rises again. If we let the system completely relax, the pressure will reach the previous plateau value π∞. Then, the monolayer is recompressed up to the area per molecule at which the detergent injection was done. If we stop compression, the surface pressure decreases down and would reach again the previous final value π∞ obtained at the end of the evolution depicted in the inset. The monolayer is compressed up to the collapse. At 0.5 nm2 a surface pressure plateau appears on the isotherm located at 43 ( 1 mN‚m-1. This surface pressure is equal to the surface pressure of the free surface of detergent solution without phospholipid monolayer. Finally, at 0.42 nm2 the surface pressure rises sharply and the monolayer collapses at 0.40 nm2, almost at the same phospholipids surface density as that on pure water. (32) Kaganer, V. M.; Mo¨hwald, H.; Dutta, P. ReV. Mod. Phys. 1999, 71, 779.

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The evolution of diffraction spectra for DMPE monolayer during the surface pressure evolution followed by the injection of Triton-X100 in the subphase is depicted in Figure 1B. The time evolutions of peak positions, fwhm, and peak intensity are depicted in Figure 1C. Initially, at 12 mN‚m-1, the monolayer exhibits two diffraction peaks as shown by the bottom curve of Figure 1B at Qxy ) 14.41 nm-1, Qz ) 5.1 nm-1 out of the plane for the 1h1 peak and Qxy ) 14.78 nm-1, Qz ) 0 nm-1 in the plane for the 02 peak. This corresponds to the classical rectangular NN-tilted chain (L2) structure of DMPE on pure water at this intermediate surface pressure. The parameters of the rectangular cell deduced from diffraction peaks are a ) 0.51 nm and b ) 0.85 nm, the area of the cell is 0.43 nm2, and the tilt of the chains is t ) 22.4°. After introduction of the detergent in the subphase, these two peaks shift as shown in Figure 1B. At the end of the evolution, only a single peak is measured in the spectrum located in plane at (Qxy ) 14.99 nm-1, Qz ) 0 nm-1). This peak corresponds to a perfectly hexagonal untilted structure of the chains (LS). The parameters of the associated rectangular cell are a ) 0.48 nm and b ) 0.84 nm, the area of the rectangular cell 0.40 nm2, and the tilt of the chains t ) 0°. Such a structure represents the more condensed organization for the DMPE molecules in monolayers and is usually measured at high pressure on pure water subphase (π > 30 mN‚m-1). The time evolution of the diffraction pattern is thus characterized by the compression and phase transition of the structure of the DMPE monolayer to high-pressure organization, which remains stable with time and with compression, and decompression of the monolayer. This structure corresponds to the one of phospholipid monolayers on pure water at the same surface pressure.24 The same experiment was performed starting with π0 ) 4 mN‚m-1, in the LE phase, and has given the same results, except the absence of diffraction peaks at t ) 0 as expected. 3.2. Detergent Concentration above the cmc. Figure 2A shows the surface pressure behavior of DMPE monolayers, initially at (π0 ) 12 mN‚m-1) in the LC phase and after the injection of Triton-X100 in the subphase at concentration c ) 0.5 mmol‚L-1. The full line at low surface pressure and high area per molecule is the compression of the monolayer up to π ) 12 mN‚m-1. The water subphase is then exchanged with the TritonX100 solution. The evolution of surface pressure with time is depicted in the inset of Figure 2A. It shows a rapid increase of surface pressure up to a plateau value π∞ ) 43 ( 0.5 mN‚m-1, equal to the surface pressure of the free surface of the detergent solution. This equilibrium pressure is reached in less than 2 h. Following a first protocol (sequence 1-2-3), the monolayer is directly compressed (3) up to the collapse of the monolayer after the end of the surface pressure time evolution. Upon compression, the surface pressure remains constant up to 0.5 nm2‚molecule-1, where the pressure starts to rise. When this compression curve reaches the isotherm curve on pure water, the compression follows the pure water curve up to the collapse which occurs at the same area per molecule (0.4 nm2) and at the same surface pressure (60 mN‚m-1). Following a second protocol (sequence 1-2-4-5), the monolayer is expanded to larger area per molecule (4), as depicted in Figure 2A. The surface pressure remains constant at π∞ up to the complete expansion where the pressure remains stable. Then, the monolayer is compressed again (5). The surface pressure remains stable upon compression up to 0.38 nm2 when it starts to rise. No evolution of surface pressure occurs if the compression is stopped. The collapse occurs at an area of 0.30 nm2 and a surface pressure of 60 ( 1 mN‚m-1. One notices that the collapse

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Fontaine et al.

Figure 1. (A) Surface pressure measurements on DMPE monolayers during and after the penetration of the monolayer by Triton-X100 with the LC (π ) 12 mN‚m-1) phase as the initial state. In the main graph are depicted the isotherms of a DMPE monolayer before and after the injection of Triton-X100 at concentration c ) 0.005 mmol‚L-1 (below the cmc) in the water subphase; the dashed curve corresponds to the π-A isotherm (compression only) of DMPE monolayer on pure water; the plain line (1) at low surface pressure and high area per molecule corresponds to the compression of the monolayer on pure water subphase; the curve at higher surface pressure corresponds to the expansion (3) and recompression (4) to collapse of the monolayer. The inset represents the time evolution of surface pressure after injection of detergent in the water subphase. (B) Evolution of the diffraction patterns with time (from bottom to top) of a DMPE monolayer initially in the LC phase, before and after the injection of Triton-X100 at concentration c ) 0.005 mmol‚L-1 (below the cmc). (C) Evolution of the peaks positions, fwhm, and total peak intensity with time after the injection of Triton-X100.

occurs at the same surface pressure but at smaller area per molecule (0.30 nm2) than monolayers on pure water surface (0.40 nm2). The evolution of diffraction peaks for DMPE monolayers during the surface pressure evolution followed by the injection of Triton-X100 in the subphase is depicted in Figure 2B. The time evolution of peak position, fwhm, and total peak intensity are depicted in Figure 2C. Initially, at 12 mN‚m-1, the structure of the monolayer is NN tilted (L2). The second peak out of plane was not measured due to the scattering by the water meniscus at high Qz values. Introducing the detergent in the subphase, this peak very slightly shifts as shown in Figure 2B. At the end of

the evolution, one single peak is measured at Qxy ) 14.98 nm-1 and Qz ) 0 nm-1, indicating a transition to a hexagonal structure. Indeed, the same position is observed for hexagonal untilted structure (LS) and on pure water. The rectangular cell parameters are a ) 0.48 nm and b ) 0.84 nm and the area 0.40 nm2. This is the more condensed organization for DMPE monolayers, usually measured at high pressure (π > 30 mN‚m-1). The time evolution of the diffraction pattern is thus characterized by the fast compression and phase transition of the structure of the DMPE monolayer to high-pressure organization, which remains stable with time.

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Figure 2. (A) Surface pressure measurements on DMPE monolayers during and after the penetration of the monolayer by Triton-X100 with the LC (π ) 12 mN‚m-1) phase as the initial state. In the main graph are depicted the isotherms of a DMPE monolayer before and after the injection of Triton-X100 at concentration c ) 0.5 mmol‚L-1 (above the cmc) in the water subphase; the dashed curve corresponds to the π-A isotherm (compression only) of DMPE monolayer on pure water; the plain line (1) at low surface pressure and high area per molecule corresponds to the compression of the monolayer on pure water subphase; the curves at higher surface pressure corresponds to a single compression (without expansion) to collapse of the monolayer (3). The other curves correspond to expansion (4) and recompression (5) to collapse of the monolayer after the end of surface pressure evolution. The inset represents the time evolution of surface pressure after injection of detergent in the water subphase. (B): Evolution of the diffraction patterns with time (from bottom to top) of a DMPE monolayer initially in the LC phase (π ) 12 mN‚m-1), before and after the injection of Triton-X100 at concentration c ) 0.5 mmol‚L-1 (above the cmc). (C) Evolution of the peak positions, fwhm, and total peak intensity with time after the injection of Triton-X100. (D) Integrated in-plane diffraction spectrum measured on a DMPE monolayer initially in the LC phase after the injection of Triton-X100 (end of surface pressure evolution) at concentration c ) 0.5 mmol‚L-1 (above the cmc). The squares are the diffraction spectrum measured at the end of surface pressure evolution following the injection of Triton-X100. The triangles are the diffraction spectrum measured at the same are per molecule but after an expansion-compression cycle. The lines represent the best fits obtained with a Lorentzian fit function and a sloping background.

In Figure 2D is depicted the diffraction peaks obtained at the end of the time evolution of surface pressure (last spectrum of Figure 2C), and the peaks obtained after expansion and recompression at the same area per molecule of the monolayer. Although the peak’s position and fwhm are identical, the intensities of the peaks are strongly different. Before the expansion, the intensity is 5.7 × 10-4. After the recompression, the intensity is 2 × 10-5. This shows that the expansion recompression cycle leads to a decrease of the amount of organized matter of factor 30 within the monolayer. The same experiment was performed starting with π0 ) 4 mN‚m-1, in the LE phase and has given the same results except

that initially no diffraction peaks were observed at t ) 0. The diffraction peaks appears with the surface pressure increase. The final state of the system exhibits the same behavior.

4. Analysis and Discussion 4.1. Surface Pressure Evolution. The common feature of these experiments performed by injecting in the subphase of a phospholipid monolayer a neutral detergent (Triton X100) is the final surface pressure reached after the end of the surface pressure time evolution π∞. Whatever the initial surface pressure of the monolayer, the final surface pressure is equal to that of the free detergent solution surface at the same concentration (πX100),

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provided π0 is below the surface pressure of the detergent πX100. Below the cmc, the final surface pressure is defined by the detergent concentration independently of the presence of the phospholipids. Above the cmc, the surface pressure equals 43 mN‚m-1, its value for a free surface solution. This means that the final surface pressure of the system is only determined by the concentration of the detergent in the subphase. Indeed, according to Gibbs law of surfactant,33,34 detergent molecules adsorb at the free surface of the solution in order to reach the equilibrium pressure fixed by the detergent concentration. According to the theory of Sundaram et al.,6 the fact that the final surface pressure is the same with phospholipid at the interface or not shows that the interaction between the phospholipid and the detergent is very weak. Among all the performed experiments, two cases did not follow this picture. If the pressure determined by the detergent concentration is above the collapse surface pressure of the monolayer (πc), the detergent adsorption leads to the collapse of the phospholipids layer. The other exception occurs when the monolayer initial pressure is above the equilibrium surface pressure of the detergent solution. Then, the surface could no more be considered as a free surface where the detergent adsorption is possible. These cases are not presented here.35 The evolution of the surface pressure upon expansion or compression could be explained considering detergent adsorption kinetics effects. Below the cmc, upon compression, the desorption rate is not sufficient to maintain the pressure constant with respect to the compression/decompression speed. Moreover, due to the area occupied by the adsorbed detergent molecules, the measured area per molecule (ratio between the experimental layer area and the number of spread phospholipid molecules) is larger than the real, microscopic area per phospholipid molecule. The surface pressure appears higher than expected at such an area. The collapse area could be then larger than for a pure phospholipids monolayer. Above the cmc, detergent desorption is a faster process. Indeed, the characteristic diffusion time scale is inversely proportional to the square of the bulk concentration.6 Thus, the surface pressure remains constant up to area per molecule where it starts to rise and where no more detergent molecules remain at the interface. However, spurious oscillations observed below the cmc indicate the convection process would not have to be neglected in this case. 4.2. Surface Behavior at the Molecular Level. The increase of surface pressure due to detergent adsorption is also revealed by the GIXD results. Indeed, whatever the initial surface pressure and the final detergent concentration, the evolution of the diffraction spectra reveals a compression of the monolayer from a lower density phase (LE or L2-phase with a large tilt of the molecule) to a higher density phase (L2-phase with smaller tilt of molecules or hexagonal phase, LS). The final structure depends on the surface pressure π∞ reached at the end of the evolution and corresponds to the structure obtained at the same surface pressure on pure water at π∞. The Gibbs law explains most of the features related to the interaction between the volume and the surface, where the two surfactant molecules are not distinguished. However, the presence of pure phospholipid domains demonstrated by GIXD is not accounted by this approach except that the compression of the domains is due to the surface pressure increase explained by Gibbs law. If the two species at the interface were effectively (33) Addamson, A. W.; Gast, A. P. Physical Chemistry of Surfaces, 6th ed.; J. Wiley and Sons: New York, 1997. (34) Israelachvili, J. Intermolecular & Surface Forces, 2nd ed.; Academic Press: London, 1992. (35) Faure´, M. C.; Fontaine, P.; Goldmann M. Unpublished results.

Fontaine et al.

equivalent, one would have expected total mixing and no organization (no diffraction peaks). However, segregation occurs since pure organized phospholipid domains are detected. Thus, it appears that the surface monolayer behaves as a Langmuir monolayer on pure water made of two different molecules except that the surface pressure is no more controlled by the accessible area for the monolayer. At the interface, the two types of molecules show their differences leading to segregation between pure phospholipids domains and detergent phase. One may notice that some phospholipids could have been dissolved at 2D in the detergent domains. The interaction between volume and surface only imposes the surface pressure on the phospholipid domains. This situation remains stable since no time evolution of the peaks parameters, especially of the peaks intensity, is detected after the end of the surface pressure evolution. The role of the detergent is to impose the surface pressure, compressing the phospholipids domains and imposing then their microscopic structure. 4.3. 2D and 3D Dissolution of the Phospholipids by Detergent. Above or below the cmc, if compression to collapse of the monolayer takes place after the adsorption of the detergent molecules at the air-water interface (without expansion, even a long time after the end of surface pressure evolution) the collapse area is identical or larger than the one on pure water. Thus, there is apparently no dissolution of molecules from the interface to the subphase. GIXD confirms this assertion since there is no evolution of peak intensity after the end of the evolution of surface pressure. Thus condensed phospholipids domains appear not to be solubilized by detergent at the interface and/or in the water subphase. Such phenomenon was not expected above the cmc since the amphiphilic environment of detergent micelles should favor phospholipids dissolution in the volume. However, this resistance is effective only if the monolayer remains without thermodynamical changes. Indeed, experiments above the cmc following the second experimental cycle in Figure 2A (1-2-4-5), exhibit a completely different behavior. Although the expansionrecompression cycle up to the initial area does not apparently change the state of the interface (no evolution of surface pressure), further compression to collapse and GIXD data reveal interesting features. The collapse area is much smaller than on pure water (0.3 compared to 0.4 nm2). This suggests that phospholipid molecules have disappeared from the monolayer, probably dissolved in the subphase by the detergent micelles. From collapse areas, this loss can be estimated to 30%. Moreover, as shown by Figure 2D, the total intensity of the diffraction peaks of the organized DMPE domains is decreased by a factor 30 after the expansion and recompression cycle (4-5), indicating less diffracting matter below the X-ray footprint (150 × 5 mm2). This is in agreement with the dissolution of phospholipid molecules by the detergent either at 3D (dissolution in the subphase) and/or at 2D (phospholipids molecules remain at the interface but mixed with detergent). Compression of the monolayer shows that the number of phospholipids molecules at the interface has decreased (smaller collapse area per DMPE molecules). Thus, the expansion of the monolayer induces dissolution in the subphase (3D) by detergent micelles. 4.4. Discussion. Not only these pure phospholipid domains are stable against 2D mixing with detergent, but they are also resistant to phospholipid dissolution by detergent micelles, provided no expansion of the monolayer is performed. This striking feature could be due to a high cohesive energy barrier to overcome for taking a molecule from a dense domain to a micelle in the subphase. The fact that this resistance is suppressed (or partially suppressed) upon expansion of the monolayer can

Unexpected Stability of Phospholipid Langmuir Monolayers

be explained first by an increase of the adsorbed detergent amount, second by the further adsorption of detergent molecules to keep constant the pressure which could lead to transient adsorption of detergent micelles at the interface which can bring phospholipid molecules during their “redissolution”. One must notice that the barrier is not completely overcome since even after a long time, phospholipid molecules remain at the interface. Such resistance to 2D and 3D dissolution of phospholipids differs from the 3D case where detergent molecules and phospholipids are both dissolved in water, the latter forming self-assembled vesicles. The mixing of detergents and phospholipids in this kind of membrane is possible.1,4 This could be explained by the fact that in monolayers, the phospholipids can be organized in a condensed phase (high pressure), which appears resistant to the detergent. In vesicles, the lipid chains are in a liquid, low-density and pressure state (the surface pressure in a membrane is believed to be zero). The lateral interaction between phospholipids within the vesicle membrane could be weaker enough to enable the intimate mixture of detergent and phospholipids and the dissolution of phospholipids in detergent micelles. Such weak interaction between DMPE and Triton X100 is also revealed by the absence of difference in the final surface pressure with and without phospholipid on top of the detergent solution. However, resistance to solubilization of lipids by Triton X100 was already detected in the case of bilayers made of mixtures of lipids5 with liposomes or supported. In the case of bilayers made of mixture of DPPC and DOPC, it was shown that DOPC domains are quickly dissolved when in contact with Triton X-100, as DPPC domains resists to solubilization a few hours. More interesting is their slow dissolution mechanism. Indeed, it appears that the boundaries of the DPPC domains remain unchanged and that the dissolution occurs through holes appearing inside the domains. This mechanism could be relevant to explain our experiment since at the end of the detergent adsorption, the domains remain unchanged showing that the detergent is not able to dissolve at 2D the phospholipid domains. However, upon expansion of the monolayer, holes can appear in the domains enabling the dissolution of phospholipids. As an explanation to the resistance to dissolution, Morandat et al. claim that it could be due to the high melting temperature of the lipids or to the molecular packing.5 Our experiments show that, effectively, the molecular packing of lipid chains is able to induce resistance of DPPC domains to dissolution both in mono- or bilayers. For the membrane protein crystallization process, the resistance of the phospholipid domains favors the 2D crystallization of

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proteins since not all the ligand molecules will be dissolved. However, the compression of the phospholipid domains imposed by the detergent could hinder the 2D diffusion of molecules at the interface which is mandatory for the crystallization process. Such a phenomenon could explain the irreproducibility of the 2D-membrane protein crystallization process since biologically relevant phospholipids exhibit the same behavior. As a consequence, one should recommend the use of a low surface pressure detergent for such procedure.

Conclusion We observed for the first time the surface behavior of phospholipids Langmuir film on a detergent solution simultaneously at macroscopic and microscopic length scales. Our results show that although the Gibbs law is the relevant scheme to interpret and predict thermodynamically the relationship between the volume and the surface, GIXD revealed unpredicted features concerning the surface behavior of the detergent-phospholipid film. Detergent effectively adsorbs at the solution-air interface in the amount needed to reach the surface pressure of detergent solution. From a macroscopic point of view, the state and chemical nature of phospholipid and detergent molecules have no influence on the final surface pressure reached. However, macroscopic surface pressure measurements do not give any information about the nature of the surface. On the other hand, GIXD revealed that adsorbed detergent and phospholipid molecules segregate in pure phospholipids domains surrounded by a detergent rich phase. The surface pressure imposed by the detergent on the interface leads to the compression of these domains. This compression results in the transition to a high-pressure state of the phospholipids domains (collapse, condensed phase, etc.). No dissolution of insoluble phospholipid molecules occurs below the cmc. However, even above the cmc, the phospholipids domains appear to resist to solubilization by the detergent micelles in the subphase provided no expansion of the monolayer is performed. Expansion of the monolayer suppresses this resistance and leads to phospholipids dissolution. Acknowledgment. We thank the BM32 and CEA/DRFMC/ SI3M technical staff for help and assistance for the installation of the dedicated Langmuir trough on the GMT instrument at the ESRF. We also thank Fre´de´ric Ne´, of the CEA/DRFMC for help and assistance during the experiments We thank D. Le´vy of the Curie Institute (Paris) for stimulating discussion. LA701293N