Assembly of Purple Membranes on Polyelectrolyte Films - Langmuir

Mar 24, 2009 - The membrane protein bacteriorhodopsin in its native membrane bound form (purple membrane) was adsorbed and incorporated into ...
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Assembly of Purple Membranes on Polyelectrolyte Films Marie-belle Saab,† Elias Estephan,† Thierry Cloitre,† Rene Legros,† Frederic J. G. Cuisinier,‡ Laszlo Zimanyi,‡,§ and Csilla Gergely*,† †

Groupe d’Etude des Semi-conducteurs, UMR 5650, CNRS-Universit e Montpellier II, 34095, Montpellier Cedex 5, France, ‡EA 4203, UFR Odontologie, Universit e Montpellier I, 34193 Montpellier Cedex 5, France, and §Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged, Hungary Received September 22, 2008. Revised Manuscript Received February 9, 2009 The membrane protein bacteriorhodopsin in its native membrane bound form (purple membrane) was adsorbed and incorporated into polyelectrolyte multilayered films, and adsorption was in situ monitored by optical waveguide lightmode spectroscopy. The formation of a single layer or a double layer of purple membranes was observed when adsorbed on negatively or positively charged surfaces, respectively. The purple membrane patches adsorbed on the polyelectrolyte multilayers were also evidenced by atomic force microscopy images. The driving forces of the adsorption process were evaluated by varying the ionic strength of the solution as well as the purple membrane concentration. At high purple membrane concentration, interpenetrating polyelectrolyte loops might provide new binding sites for the adsorption of a second layer of purple membranes, whereas at lower concentrations only a single layer is formed. Negative surfaces do not promote a second protein layer adsorption. Driving forces other than just electrostatic ones, such as hydrophobic forces, should play a role in the polyelectrolyte/purple membrane layering. The subtle interplay of all these factors determines the formation of the polyelectrolyte/purple membrane matrix with a presumably high degree of orientation for the incorporated purple membranes, with their cytoplasmic, or extracellular side toward the bulk on negatively or positively charged polyelectrolyte, respectively. The structural stability of bacteriorhodopsin during adsorption onto the surface and incorporation into the polyelectrolyte multilayers was investigated by Fourier transform infrared spectroscopy in attenuated total reflection mode. Adsorption and incorporation of purple membranes within polyelectrolyte multilayers does not disturb the conformational majority of membrane-embedded R-helix structures of the protein, but may slightly alter the structure of the extramembraneous segments or their interaction with the environment. This high stability is different from the lower stability of the predominantly β-sheet structures of numerous globular proteins when adsorbed onto surfaces.

Introduction The interactions between biological macromolecules and solid surfaces are of primary importance in numerous fields of material sciences and in the design of new coatings for biomaterials. Protein adsorption on surfaces is a complex process, which is governed by the hydrophobicity and hydrophilicity of the surface,1 the surface charge,2 roughness,3 and free energy.4,5 Direct adsorption of a protein onto a surface often induces loss of the functional activity and denaturing of the adsorbed protein. The use of underlying polyelectrolyte films (PEFs) may offer convenient solutions for the problems encountered due to direct anchoring of proteins to bare surfaces. PEFs are built up by the alternated adsorption of polycations and polyanions from aqueous solution at a solid/liquid interface.6,7 Due to this layer-bylayer construction, all PEFs exhibit excess charges, alternatively positive and negative, on their surfaces. These excess charges are *Corresponding author. Address: Groupe d’Etude des Semiconducteurs, UMR 5650, CNRS-Universite Montpellier II, Pl. Eugene Bataillon 34095 Montpellier Cedex 5, France. Tel: 0033467143248. Fax: 0033467143760. E-mail: [email protected]. (1) Grinnell, F.; Feld, M. K. J. Biol. Chem. 1982, 257, 4888–4893. (2) Qiu, Q.; Sayer, M.; Kawaja, M.; Shen, X.; Davies, J. E. J. Biomed. Mater. Res. 1998, 42, 117–127. (3) Dufrene, Y. F.; Marchal, T. G.; Rouxhet, P. G. Langmuir 1999, 15, 2871– 2878. (4) Absolom, D. R.; Zing, W.; Neumann, A. W. J. Biomed. Mater. Res. 1987, 21, 161–171. (5) Norde, W.; Lyklema, J. Biomater. Sci. Polym. Ed. 2 1991, 183-202. (6) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 210, 831–835. (7) Decher, G. Science 1997, 277, 1232–1237.

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the motor of their buildup, and also facilitate the adsorption of a great variety of compounds on the PEF surface.8-10 Previously, it was shown that proteins may adsorb on either positively or negatively charged polyelectrolyte (PE)-terminating films.11,12 However, the ionic strength, pH, and the charge difference between the adsorbed protein and terminating film layer influence the thickness and the amount of protein taken up. These physicochemical parameters provide a fine-tuning in the properties of the obtained multilayered films. A PEF coating can also preserve the secondary structure of adsorbed proteins.13,14 Therefore, optimization and characterization of the applied PEFs are essential for each application. It is certainly due to their numerous tunable properties that the use of various multilayered PEFs has been greatly extended in biomaterials science. The aim of our work is to use PEFs as a scaffold to produce an ordered matrix incorporating membrane proteins in an oriented way within the PE layers. To this end, the adsorption of bacteriorhodopsin (BR) in its membrane-bound form (purple (8) Picart, C.; Lavalle, P.; Hubert, P.; Cuisinier, F. J. G.; Decher, G.; Schaaf, P.; Voegel, J.-C. Langmuir 2001, 17, 7414–7424. (9) Gergely, C.; Bahi, S.; Szalontai, B.; Flores, H.; Schaaf, P.; Voegel, J.-C.; Cuisinier, F. J. G. Langmuir 2004, 20, 5575–5582. (10) Gergely, C.; Szalontai, B.; Moradian-Oldak, J.; Cuisinier, F. J. G. Biomacromolecules 2007, 8, 2228–2236. (11) Ladam, G.; Gergely, C.; Senger, B.; Decher, G.; Voegel, J.-C.; Schaaf, P.; Cuisinier, F. J. G. Biomacromolecules 2000, 1, 674–687. (12) Picart, C.; Ladam, G.; Senger, B.; Voegel, J.-C.; Schaaf, P.; Cuisinier, F. J. G.; Gergely, C. J. Chem. Phys. 2001, 115, 1086–1094. (13) Caruso, F.; Mohwald, H. J. Am. Chem. Soc. 1999, 121, 6039–6046. (14) Schwinte, P.; Voegel, J.-C.; Picart, C.; Haikel, Y.; Schaaf, P.; Szalontai, B. J. Phys. Chem. B. 2001, 105, 11906–11916.

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membrane, PM) on charged surfaces of PEs was studied under different physicochemical conditions in order to explore the various interactions at the base of the adsorption of membrane proteins. PM patches are localized in the cell membrane of Halobacterium salinarum, and consist of the sole protein BR and lipids in a 75-25% (w/w) composition.15 BR is a seven-transmembrane Rhelical protein with a covalently bound retinal chromophore. Its function is light-driven transmembrane proton pumping against the proton electrochemical gradient across the cell membrane, and thereby conserving free energy for ATP synthesis. The X-ray structure of BR is known at atomic resolution,16 and most of the molecular details of its proton transporting photocycle have been elucidated. BR is also one of the most promising candidates both in solubilized form and in PM for biomaterial sciences applications because of its enormous stability and favorable optical properties. Because of its unique optical properties, numerous applications using BR as an integrated optical switch are also forecasted. PM possesses a permanent electric dipole moment17 and therefore can be oriented in an electric field. The static and the dynamic response of optical waveguides coated with a dried PM film have been reported previously.18-20 In these works, PM was deposited by evaporation of water from the protein solution, thus no control over the thickness or orientation was assured, and inhomogeneities in the thickness of the obtained BR film were observed.20 Transient absorption and photovoltage studies of self-assembled PM/polycation multilayer films have been previously reported,21 but without the aim of structural and optical characterization of the obtained architectures. In this sense, new solutions for layering BR in its membrane bound form in a controlled way, onto any substrate, as well as incorporating them in a matrix with a high local organization, could be of wide interest. Here we report on the adsorption of PMs on and into PE multilayered films produced by the deposition of polyethyleneimine (PEI) followed by the alternating physisorption of anionic polystyrene sulfonate (PSS) and cationic polyallylamine (PAH). The step-by-step buildup of the multilayers and the PM adsorption were monitored in terms of the thickness and the refractive index by means of optical waveguide light-mode spectroscopy (OWLS). No changes in the secondary structure of the membrane-bound BR as a consequence of its insertion into PEF architectures were observed by Fourier transform infrared spectroscopy in attenuated total reflection mode (FTIR-ATR). Atomic force microscopy (AFM) was employed to visualize the surface of the PEFs and the layered PMs. These detailed pieces of structural information on the PE/BR matrices could be interesting in further photoelectric and photochromic applications of this material.

Materials and Methods Materials. Anionic poly(sodium 4-styrenesulfonate) (PSS, MW = 60 000), cationic poly(allylamine hydrochloride) (PAH, (15) Oesterhelt, D.; Stoeckenius, W. Methods Enzymol. 1974, XXXI, 667–678. (16) H. Luecke, B.; Schobert, H. T.; Richter, J.; Cartailler, P.; Lanyi, J. K. J. Mol. Biol. 1999, 291, 899–911. (17) Barabas, K.; Der, A.; Dancshazy, Zs.; Ormos, P.; Keszthelyi, L; Marden, M. Biophys. J. 1983, 43, 5–11. (18) Ormos, P.; Fabian, L.; Oroszi, L.; Wolff, E. K.; Ramsden, J. J.; Der, A. Appl. Phys. Lett. 2002, 80, 4060–4062. (19) Fabian, L.; Oroszi, L.; Ormos, P.; Der, A. In Molecular Electronics: Biosensors and Bio-computers; Barsanti, L. Ed.; Kluwer Academic Publishers: Dordrecht/Boston/London, 2003; p 341. (20) Lukacs, A.; Garab, G.; Papp, E. Biosens. Bioelectron. 2006, 21, 1606–1612. (21) Jussila, T.; Li, M.; Tkachenko, N. V.; Parkkinen, S.; Li, B.; Jiang, L.; Lemmetynien, H. Biosens. Bioelectron. 2002, 17, 509–515.

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MW = 70 000), and cationic poly(ethyleneimine) (PEI, MW = 60 000) were purchased from Aldrich. NaCl (purity 99.5%) was purchased from Fluka; tris-(hydroxymethyl)-aminomethane (TRIS) and 2-(N-morpholino)ethanesulfonic acid (MES) were from Sigma. All the chemicals of commercial origin were used without further purification. Ultrapure water (Milli-Q plus system, Millipore) was used for solutions and in the different cleaning steps. All buffer solutions were degassed under vacuum and filtered before use. The PEs were dissolved in 25 mM MES, 25 mM TRIS, and 100 mM NaCl (pH 7.4) buffer (0.15 M ionic strength) at a concentration of 5 mg/mL. Some experiments were performed at three different ionic strengths (0.05, 0.15 and 0.55 M) by varying the concentration of NaCl in the buffer (0, 0.1, and 0.5 M, respectively). PMs were isolated from Halobacterium salinarum following standard procedures.13 PM were suspended in the same buffer as the PEs. The PM concentration is referred to by the corresponding protein concentrations of 30 μM and 150 μM, determined using the visible and near UV extinctions of BR. PEFs were built up by sequential physisorption of PSS and PAH onto a precursor layer of PEI, and all adsorption steps were separated by rinsing. Experimental Methods. Optical Waveguide LightMode Spectroscopy. The buildup of multilayered PEFs and protein adsorption onto PEFs were followed in situ by OWLS, an optical technique allowing in situ study of the adsorption of macromolecules onto a substrate.22 The technique is based on the confinement of a laser beam in a planar waveguide formed by a high refractive index layer (n ∼ 1.7) coated onto a glass substrate.22,23 For a discrete coupling angle, the laser beam is coupled into the waveguide via a diffraction grating and the propagation of the totally reflected light is monitored in real time. When material adsorbs, it perturbs the evanescent field and leads to changes in the effective refractive index of the guided transverse electric (NTE) and magnetic (NTM) modes, highly sensitive to the changes of the refractive indices. OWLS records variations in NTE and NTM with high precision (ΔN ∼ 10-5) roughly up to a film thickness of 350 nm, corresponding to half of the wavelength of the laser.24 By measuring the two modes simultaneously, then solving the mode equations,12,24 the structural parameters of the adsorbed layers, i.e., refractive index and thickness (nA,dA) were obtained. OWLS data were analyzed by assuming that the multilayers behave as homogeneous and isotropic films. Anisotropy cannot be excluded for dry and/or thin films as recently reported.25 All our experiments were performed in a liquid cell, and the rather thick PEFs were built up from aqueous solutions. Earlier scanning angle reflectometry measurements on PSS-PAH wet films were successfully analyzed by the optical invariants method and evidenced that these layers can be considered homogeneous and isotropic.12,24 The buildup of the PE multilayer film was performed as follows. First, the PEI solution (5 mg/mL) was injected into the cell and left to adsorb for 20 min. Then, in the same way, architectures of PEI-PSS, PEI-PSS-PAH, and PEI-(PSSPAH)2 were built progressively. PE adsorption was always separated by a 20 min long rinsing step to remove excess material from the measuring cell. PMs were then adsorbed onto these multilayer films and allowed to adsorb from a continuous flow through the measuring cell (4 mL/h) for about 1 h. Once the optical signals leveled off, the protein solution was replaced by buffer solution and desorption of the adsorbed protein (if any) was monitored. The experiments (both PEF construction and PM adsorption) were performed at various ionic strengths using three different concentrations of NaCl: 0.05 M buffer (0 M NaCl, 0.025 M MES, 0.025 M TRIS), 0.15 M buffer (0.1 M NaCl, 0.025 M MES, 0.025 M TRIS), and 0.55 M buffer (0.5 M (22) Tienfenthaler, K.; Lukosz, W. J. Opt. Soc. Am. B 1989, 6, 209–215. (23) Ramsden, J. J. J. Mol. Recogn. 1997, 10, 109–120. (24) Picart, C.; Gergely, C.; Arntz, Y.; Voegel, J.-C.; Schaaf, P.; Cuisinier, F. J. G.; Senger, B. Biosens. Bioelectron. 2004, 20, 553–561. (25) Horvath, R.; Ramsden, J. J. Langmuir 2007, 23, 9330–9334.

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NaCl, 0.025 M MES, 0.025 M TRIS). PM was adsorbed onto either positive (PAH) or negative (PSS) PE-terminated films. After the last PM adsorption step, the buildup of PE layers with alternating charge was continued by first applying the same PE as the last one before PM. Atomic Force Microscopy. AFM images of the PE/protein layers were recorded in buffer, with an Asylum MFP-3D head and Molecular Force Probe 3D controller (Asylum Research, Santa Barbara, CA). Height and phase images were taken in tapping mode using silicon nitride and rectangular cantilevers (Olympus Microcantilever, OMCL-BL-RC150VB) at a drive frequency of 18 KHz; only the height images are reported. We always performed several scans over a given surface area assuring reproducible images. Typically 512  512 point scans were taken at 1 Hz scan rate. Images were first recorded on 20  20 μm2, or 15  15 μm2 scan size, then on 5  5 μm2, 1.5  1.5 μm2 and 1  1 μm2 for a better resolution. The PEF and then the PMs were layered and left to adsorb on a mica substrate from the same solutions as for the OWLS experiments. Profilometric section analysis allowed locating the PM patches on top of the PEF. Fourier Transform Infrared Spectroscopy in Attenuated Total Reflection Mode. The structural changes of the adsorbed PE multilayers and incorporated BR during the construction of PEI-PSS-PAH-PM-PSS-PAH were followed by recording the FTIR-ATR spectra with a Bruker IFS 66V spectrophotometer. In these experiments, the PEFs were built on the ZnSe crystal of the fluid ATR cell, and the intensity of the evanescent wave propagating at the crystal/multilayer interface was measured by a deuterated triglycide sulfate (DTGS) detector and aperture-type KBr, and cooled to the temperature of liquid nitrogen. The multilayers were constructed in situ on the ATR crystal by successive adsorption of PEs diluted in the same buffer: 25 mM MES, 25 mM TRIS, 100 mM NaCl, pH 7.4. As H2O presents a strong absorption in the infrared range also characteristic of proteins, D2O was used instead in all dilutions. The PEs and the PM suspension were circulated with a peristaltic pump above the ZnSe crystal (in a closed circuit) at a 0.5 mL/min flow rate until the adsorbed amount reached saturation. After each adsorption, the cell was emptied and rinsed by the buffer solution for about 10 min, thus the multilayers were constructed in the same way as in OWLS. A spectrum resulting from the accumulation of 100 interferograms was acquired after each adsorption stage with a resolution of 2 cm-1. The buffer spectrum was taken as reference.

Results and Discussion Adsorption Kinetics of the PE/Protein Layers Monitored In Situ by OWLS. The adsorption of PM at 0.15 M ionic strength was performed on positively charged films ending with PAH, and negatively charged films ending with PSS. The two corresponding types of construction were PEI-(PSS-PAH)2PMxn-PAH-PSS-PAH and PEI-(PSS-PAH)2-PSSPMxn-PSS-PAH-PSS (where n = 1-4, the number of adsorption steps). The effect of the membrane concentration on the construction of the PEF/PM matrix was also studied. The recorded changes in the effective refractive index of the transverse electric mode (NTE) versus time indicate the step by step buildup of the PEI-(PSS-PAH)2 and the PEI-(PSSPAH)2-PSS films (Figure 1, Ia and IIa, respectively) onto the planar waveguide and the following four-step adsorption of the PM at 30 and 150 μM protein concentration. Buildup was continued by adsorbing new PE layers on the top of the protein layer. The variation of the thickness of the adsorbed layers, as calculated from the NTE and NTM values, during multilayer construction is presented in Figure 1Ib,IIb. In these curves, Langmuir 2009, 25(9), 5159–5167

adsorption of PMs becomes more visible than in the measured NTE and NTM raw data, where the differences in the refractive indices (nc) of the PM suspension compared to that of the buffer (see Supporting Information) obscure the signal of adsorption. However, the insets presenting a zoom of the NTE raw data during the adsorption and rinsing steps of the PM reveal the signal variation due to the differences in the refractive indices of the PM suspension and the buffer. If we compare the adsorption of the PM on PAH and PSS ending PEFs, it is clear that adsorption of PM is possible on both the positively and negatively charged PEs, even if there are differences. Adsorption on the negatively charged PSS is rather surprising, as the surface of the PM bears mostly negative charges (isoelectric point pI = 5.2).17,26 This behavior renders it likely that other than electrostatic phenomena are also contributing to the adsorption process of the PMs on charged surfaces. At first sight, adsorption of PM exhibits relatively fast kinetics, as saturation is reached within 10-15 min. However, we have to note here that, when rinsing was performed immediately, protein was removed (data not shown). A number of experiments like those in Figure 1 let us conclude that an adsorption time of about 1 h is needed to obtain an irreversible binding of PM, presumably due to a second process of rearrangement of the PMs and/or the PEF surface. Moreover, when adsorbed on a negatively or positively charged surface, the PMs can be incorporated only by new bilayers starting by the oppositely charged PEFs. PEF layering onto the PM was possible only in this way, and no stable adsorption of PE onto the PM layer was observed when layering was continued with identically charged PEs: PSS-PM-PSS, or PAH-PM-PAH. This is demonstrated by the full removal of the layer of like charge during rinsing, but buildup of a new layer, mostly resisting rinsing from the oppositely charged PE (Figure 1, Ib and IIb). The layer-by-layer increase in the thickness (Figure 1, Ib and IIb) of the obtained architectures shows that, at low concentration (30 μM) PM interacts strongly with the positively terminating (PAH) PEF and a second injection of 150 μM concentrated PM contributes with an additional protein layer. On a 26.5 ( 1.5 nm thick positively charged PAH-ending film, the PM30 was forming a 3.5 ( 0.25 nm thick layer that grew to 11 ( 1.5 nm when a more concentrated solution of PM150 was added (Figure 1 Ib). When injected on a 29.5 ( 1.5 nm thick, negatively charged PSS-ending film, the PM30 did not adsorb, and a second injection of concentrated PM150 was needed to form a layer of 4.5 ( 0.5 nm thickness. Nevertheless, when more BR injections were carried out, no further protein adsorption was observed. Given that the PM has a dimension of 500 nm  5 nm (diameter  height),15,17 our measurements suggest that the PM formed a double-layer on the positively charged PEF, whereas on the negatively charged PEF a single-layer of PM has been deposited. As to the driving forces of this two-step adsorption process of the PM, one has to consider both its net negative electric charge and its strong permanent electric dipole moment perpendicular to the membrane plane (2.9  106 D at pH > 5).17,27 The negative charge plays an obvious role in promoting adsorption on the positively charged PEF. The fact that PM adsorption was also observed on negatively charged PEF indicates that thermodynamic contributions (presumably entropic factors due to hydration of PM and PEF) can override the electrostatic (26) Ross, P. E.; Helgerson, S. L.; Miercke, L. J.W.; Dratz, E. A. Biochim. Biophys. Acta 1989, 991, 134–140. (27) Keszthelyi, L. Biochim. Biophys. Acta 1980, 598, 429–436.

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Figure 1. Changes of the effective refractive index of the transverse electric mode (NTE) upon buildup of PEI-(PSS-PAH)2-PM30(PM150)x3-PAH-PSS-PAH and PEI-(PSS-PAH)2-PSS-PM30-(PM150)x3-PSS-PAH-PSS architectures (Ia and IIa, respectively) and the calculated film thickness (Ib, IIb). The PEI, PSS, and PAH PEs were adsorbed from a 5 mg/mL solution in 25 mM TRIS, 25 mM MES, and 100 mM NaCl (pH 7.4). PM was adsorbed from 30 and 150 μM solutions in the same buffer.

repulsion. The multilayer buildup after PM adsorption could only be continued with a layer of opposite charge, which is a strong indication of the oriented adsorption of PM as a result of its permanent dipole moment. To evidence the contribution of electrostatic interactions in the multilayer buildup, experiments at three different ionic strengths (0.05, 0.15, 0.55 M) were carried out. Figure 2 gathers the results obtained when layering PM on a PEF ending with the positively charged PAH. We found, by increasing the concentration of NaCl in the buffer, that the thickness of PEF increases showing that the dominant driving forces of these multilayer constructions of PEs are electrostatic forces. As the content of ions in the solution increases, the charges of PEs are screened, thus they build in vermicular structures resulting in large thicknesses.11 To the contrary, when the ionic strength is low, PEs use all their charges to adhere to the surface, resulting in thinner layers. The capacity of certain proteins to adsorb on either positively or negatively charged PE-terminated films has been previously described.9-12 In these works, the thickness of the protein layer and the amount of the uptaken protein varied largely with the ionic strength and with the charge difference between the adsorbed protein and the terminating film layer, pointing out the electrostatic origin of the interactions governing adsorption. Contrary to these works, we found that the total thickness of PM adsorbed on the positively (PAH) terminating multilayer 5162

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Figure 2. Comparison of layer-by-layer growth of the thickness of the PEI-(PSS-PAH)2-PM-(PSS-PAH) or PEI-(PSSPAH)2-PM-(PAH-PSS) architectures at different ionic strengths as a function of the number of layers. The buffer was 25 mM MES, 25 mM TRIS with 0, 100, and 500 mM NaCl, pH 7.4.

film is the same (10.0 ( 0.2 nm) for the three different ionic strengths. However, the presence of the PM layers does not alter the further, electrostatically driven, buildup of a new PSS-PAH Langmuir 2009, 25(9), 5159–5167

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Figure 3. Thickness of the PM layer adsorbed on PAH-ending PEFs as a function of the ionic strength of the buffer and protein concentration. Experimental conditions were the same as for Figure 2.

bilayer in the sense that the thickness of the new layers depends similarly on the ionic strength as that of the PEF below the PM layer. The construction of the multilayered PE/protein assembly may also be affected by cooperative phenomena depending on the concentration of the PM suspension in contact with the adsorption surface. Thus experiments were performed when PM was adsorbed onto the PEF in one or two steps at different concentrations. The resulting PM layer thicknesses (averages of three experiments per point) as a function of ionic strength and protein concentration are gathered in Figure 3. The thickness of the PM layer adsorbed on a positively (PAH) terminating film at a low PM concentration of 30 μM depends on the ionic strength: it is very small at a low concentration of NaCl, it passes through a maximum at the ionic strength of 0.15 M, and then decreases again at high, 0.55 M salt concentrations. This points out the electrostatic character of the interactions between the PEF and PM at low membrane concentration.28 Adsorbed at this low concentration and from a 0.15 M buffer, the obtained thickness of the PM was ∼3 nm on a positively (PAH) terminating film, suggesting an incomplete surface coverage. A second injection after rinsing at the same low concentration (PM30PM30 on PAH) contributes with some more adsorbed material, resulting in a 5nm thick PM layer that corresponds to a monolayer of 5 nm high PMs. When a second injection of more concentrated (150 μM) PM was carried out, the obtained thickness increased up to 11 nm. This corresponds to the formation of a bilayer of PMs. The formation of a second layer of PM when adsorbed on a positively terminating film was observed for all the ionic strengths. The independence of the obtained layer thickness on the ionic strength demonstrates that, at high PM concentration, other driving forces than just electrostatic ones contribute to the adsorption process. Moreover, this second class of interactions seems to promote the adsorption of two layers. The layering process saturates with the formation of a bilayer of PMs, since when 150 μM concentrated PM was adsorbed in one, or two steps, the same thickness of about 9 nm was obtained. As previously noted, a third and a fourth protein (28) Ladam, G.; Schaaf, P.; Decher, G.; Voegel, J.-C.; Cuisinier, F. J. G. Biomol. Eng. 2002, 19, 273–280.

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Figure 4. The calculated refractive indices of the adsorbed PEFPM as a function of the ionic strength and membrane concentration. Experimental conditions were the same as for Figures 2 and 3.

injection resulted in no further material adsorption. The thickness of the first PM layer deposited from the 30 μM suspension at 150 mM ionic strength on top of the PSS (negative) upper PEF surface was measured as ∼1 nm, and the total thickness after rinsing and additional deposition from the 150 μM PM suspension became ∼5 nm. The latter corresponds to a monolayer of PM on the PSS ending PEF. The calculated refractive indices of adsorbed PEF-PM layers can provide information on the hydration state of the obtained matrix, therefore we plotted them as a function of ionic strength and PM concentration (Figure 4). We can note that the refractive index of the adsorbed PEFPM layer decreases with increasing ionic strength: in the presence of ions (0.55 M), the obtained coating seems to have a more hydrated structure than at low ionic strengths (0.05 M), and this effect was already observed for other systems, too.27 A slight decrease in the refractive indices (n) with the ionic strength of thinner adsorbed PE layers has been reported previosuly.11 Our results indicate a more accentuated decrease in n that might be related to the fact that the thicker multilayered films obtained in our work can get more easily hydrated. When comparing the refractive indices of the obtained multilayers with PM adsorbed on positively (PAH) or negatively (PSS) terminating film (not shown), the same values were obtained. Thus the adsorbed PM cover layer does not influence the optical properties of the multilayer structure. This might be important for further optoelectronic applications of these combined PE/BR architectures. Thus adsorption of PMs is a complex process depending on the membrane concentration and surface charge and can be interpreted as follows: (i) for low PM concentrations, as time evolves, the PAH PE readjusts its conformation leading to a tighter interaction with PM that prevents further adsorption; (ii) for high PM concentrations, there is no time for such a readjustment and PE loops can emerge out of the first adsorbed protein layer, providing new binding sites for a second subsequent PM adsorption. The layering process is limited by the interpenetration depth of the PEs, in our case, at two layers of PM. The same effects were previously observed for other proteins adsorbed on the same type of PEF.28 However, in the case of highly concentrated PM, adsorption of a second layer must be promoted by other types of interactions (hydrophobic, DOI: 10.1021/la9002274

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Figure 5. Schematic representation of the adsorption process of the PMs on the top of the (a) PEI-(PSS-PAH)2 and (b) PEI-(PSS-

PAH)2-PSS multilayered films. Positively charged PE loops can emerge out of the first adsorbed PM layer leading to subsequent PM adsorption.

effect of hydration) to override the electrostatic repulsion between deposited PMs. This process dominates at high PM concentrations and only on positively charged (PAHterminating) surface. To the contrary, on negatively charged (PSS ending) film adsorption never exceeds the formation of a single layer of PM, probably because of the domination of the repulsive forces between the negatively charged PSS and PM. A schematic representation of the proposed adsorption process of the PM patches onto the PEFs in an optimal condition of medium ionic strength is illustrated in Figure 5. Moreover, in the two cases, the orientation of the deposited PMs is expected to be different. It is known that in neutral and alkaline environments, the cytoplasmic side of PM fragments bears more negative charges than the extracellular side, and the permanent electric dipole moment, which is parallel to the membrane normal, points toward the extracellular side.17 When adsorbed on the positive PAH, PMs should preferably face with their cytoplasmic side presenting more negative charges toward PAH. The second PM layer is then expected to take up the same orientation as a result of the interaction of the dipole moment of the approaching PMs with the electric field close to the PE surface, to the electrostatic interaction with the charges on PAH, and despite the electrostatic repulsion between the PMs. This should result in a final PM layer facing up with the extracellular side presenting less negative and more positive charges for the adsorption of a new PSS layer. Contrarily, when PAH was injected, no adsorption was observed, indicating again the extracellular orientation of the PM toward the bulk (Figure 1a,b). When adsorbed on the negatively charged PSS, PMs are expected to face it with their extracellular side presenting less negative charges. Thus, finally, a PM layer facing up with the cytoplasmic side (strongly negative) is formed, allowing further adsorption of only the positive PAH and not of the negative PSS, as it was indeed observed (Figure 1a,b). Hence, we might conclude that adsorption resulted in the formation of oriented single or double layers of PMs, facing the bulk with the cytoplasmic or the extracellular side on a negatively or positively charged surface, respectively. Morphological Studies by AFM. The morphological study of the PE/protein layers was performed by AFM leading to valuable information on the surface of the obtained matrices. First, the images of the PAH- or PSS-terminating layers of PEF, before PM injection, were recorded in liquid phase and tapping mode, revealing a rather smooth surface of a roughness of 3 nm expressed by root-mean-square. In Figure 6a, we show the surface of the PAH-terminating layer, and the AFM image of the PSS layer was very similar (not shown). The deposition of PM patches can then be noticed when PM at a concentration of 150 μM was adsorbed in one step on the positive PAH surface (Figure 6b). The corresponding images for the PM adsorbed on PSS-terminating layers are shown in the Supporting Information. A surface coverage of 63% (Figure 6b) can be calculated for the PM fragments (encircled in red) adsorbed in one step on the PAH-ending PEF. This value is around the theoretical jamming limit of 54.6% predicted by the random sequential 5164

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adsorption (RSA) theory. 29 The maximum surface coverage can slightly increase for polydisperse hard disk monolayers up to 73%. 30 Thus we may conclude that the 63% surface coverage with PMs is close to the formation of a monolayer. A second exposure to the PM suspension promoted the adsorption of new PMs, and the surface became covered at 87% (Figure 6c), which is substantially above the theoretical jamming limit. Acknowledging that the polydispersity of the PM suspension is the same, such a tight packing can only be explained if one considers that a second layer of PMs is formed. Thus, our AFM images support the results obtained with OWLS, i.e., the formation of a bilayer of PM on the PAH-terminating PEF. The profilometric sections (in the Supporting Information) taken between different points of the surface clearly indicate a thickness of about 10 nm that corresponds to a double layer of PMs. The OWLS experiments have also shown that the buildup of PEF on the top of PM is possible. Thus, AFM images were recorded to document this process as well. Indeed, the AFM image presented in Figure 6d demonstrates an almost complete coverage of the PMs by a further bilayer of PSS-PAH. When imaging at higher resolution, single PM patches can be noticed on the surface of both PEFs (Figure 7). They have a typical diameter of ∼1 μm as seen in the profilometric sections (see also Supporting Information). There are several previous studies showing AFM images (in contact mode) with subnanometer resolution recorded on PMs adsorbed on freshly cleaved mica.31,32 Our AFM images, taken in tapping mode and with a very soft cantilever, were designed to monitor the arrangement of membrane patches adsorbed onto soft PE layers, thus no better resolution could be expected from these measurements. Secondary Structure of the Protein Monitored by FTIRATR Measurements. To essay the secondary structure of BR during PM adsorption, we performed FTIR spectroscopy in ATR mode. The PEI-PSS-PAH-PM-PSS-PAH architecture was in situ built up on the ATR cell from the 0.15 M buffer but diluted in D2O. Infrared spectra were recorded after each adsorption step, thereby monitoring the layer-by-layer buildup of the PEF/PM architecture. The appearance of three major characteristic bands of the protein can be observed at 1665 cm-1,1545 cm-1, and 1455 cm-1 that correspond to the stretching vibrations of the CdO (amide I band), the combination of bending modes of -N-H and stretching vibration of -C-N (amide II band), and to the scissors vibration of the CH2, respectively (Figure 8A). The small peak around 1525 cm-1 arises from the ethylenic stretching modes of the retinal chromophore in BR.33 The secondary structure of the membrane-bound BR, incorporated into the PEF, was determined by fitting Lorentzianshaped component bands to the 1700-1600 cm-1 region of the (29) Senger, B.; Bafaluy, F. J.; Schaaf, P.; Schmitt, A.; Voegel, J.-C. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9449–9453. (30) Doty, R. C.; Bonnecaze, R. T.; Korgel, B. A. Phys.Rev. E. 2002, 65, 061503. (31) Muller, D. J.; Heymann, J. B.; Oesterhelt, F.; Moller, C.; Gaub, H.; Buldt, G.; Engel, A. Biochim. Biophys. Acta 2000, 1460, 27–28. (32) Scheuring, S.; Muller, D. J.; Stahlberg, H.; Engel, H.-A.; Engel, A. Eur. Biophys. J. 2002, 31, 172–178. (33) Marrero, H.; Rothschild, K. J. Biophys. J. 1987, 52, 629–635.

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Figure 6. Surface topography of the (a) PEI-(PSS-PAH)2 multilayered film, (b) the PMs (PM150) adsorbed on the top of a PEI-(PSSPAH)2 multilayered film in one step, (c) and that obtained in two steps, as recorded by AFM in liquid and tapping mode. (d) PM incorporation was also monitored by imaging the surface of the PEI-(PSS-PAH)2(PM150)x2-PSS-PAH multilayer. PM membrane fragments are encircled in red. Experimental conditions were the same as for the OWLS measurements; the protein was left to adsorb for 1 h from a 150 μM suspension, then rinsed.

FTIR spectra (Figure 8B). The amide I region is very complex: it contains several overlapping component bands assignable to different secondary structure elements of the protein. The number and the initial position of the component bands during fitting were set to comport with literature data and with the published atomic resolution crystal structure of BR.16,34,35 The result of the fit to the final PSS-PAH-PM-PSS-PAH structure is shown in Figure 8B . Table 1 gathers the results of the deconvolution of the amide I band, leading to a thorough structural analysis of BR when adsorbed on a PAH-ending PEF, then covered with PSS, and finally with PAH. The peak positions, amplitudes, and widths remain unchanged within error when the PMs are incorporated into the PEF by depositing further layers on the PM layer. Moreover, the secondary structural compositions obtained in our experiments are in good agreement with literature data on (34) Cladera, J.; Sabes, M.; Padros, E. Biochemistry 1992, 31, 12363–12368. (35) Cladera, J.; Torres, J.; Padros, E. Biophys. J. 1996, 70, 2882–2887.

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structural analysis based on amide I band fits.34,35 These fits uniformly show a slightly different composition of secondary structural elements than that obtained by atomic resolution X-ray diffraction, notably an underestimation of the R-helical content. However, compared to the already published results concerning the secondary structure of BR in solution, our data reveal a slight decrease in the quantity of reversed turns in favor of a β-sheet for BR in adsorbed form, certainly due to the interactions of these extra-membranous segments with the PEFs. The quantity of the most dominant structures as the R helices and disordered structure rests the same, thus we can conclude that the incorporation of PM into PEF did not alter the structure of membrane-bound BR. An important observation of the infrared studies published on BR was that the overall frequencies of the amide I and II vibrations for BR are at least 10 cm-1 higher than values found for most R-helical polypeptides and proteins.36 This has been (36) Rothschild, K. J.; Clark, N. A. Biophys. J. 1979, 25, 473–488.

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Figure 7. Surface topography and profilometric sections of the PMs (PM150) adsorbed on the top of (a) a PEI-(PSS/PAH)2 multilayered film and (b) a PEI-(PSS/PAH)2-PSS multilayered film, as recorded by AFM in liquid and tapping mode. Experimental conditions were the same as for the OWLS measurements.

Figure 8. (a) FTIR-ATR spectra monitoring the adsorption of consecutive layers of PEF starting with PEI-PSS, followed by PAH, PM PSS, and PAH. (b) Deconvolution of the amide I band of the membrane-bound BR incorporated into the PE multilayer. The experimental conditions were the same as for the OWLS experiments. Table 1. Secondary Structure Analysis of the PM Based on the Decomposition of the Amide I Region into Lorentzian-Shaped Component Bands β-sheet

disordered structures

-PAH-PM

RI helix

RII helix

reversed turns

1638.2((2.0) 1649.5((3.2) 1655.9((1.0) 1665.2((0.5) 1685.0((1.5) 17.97((4.2) 7.95((4.1) 6.26((1.5) 10.75((1.2) 14.92((1.8) 28.32 8.13 12.00 45.73 5.82 -PAH-PM-PSS 1637.8((2.6) 1649.7((4.0) 1656.1((1.3) 1665.3((0.5) 1685.3((2.0) 17.13((4.4) 8.85((8.4) 6.72((3.3) 10.82((1.4) 13.98((12.0) 27.37 9.62 11.82 45.88 5.32 -PAH-PM-PSS-PAH 1638.0((1.9) 1650.2((2.4) 1656.3((0.8) 1665.1((0.4) 1684.5((3.5) 18.14((5.9) 8.23((5.2) 5.88((2.1) 10.72((1.7) 15.31((21.5) 28.38 10.21 10.48 45.01 5.92 a F = vibration frequency in cm-1. b W = bandwidth. c % = percent of the deconvoluted band relative to the total amide I band intensity.

explained by the presence of an RII helix34,35 and an unusual primary sequence that promotes the specific interactions of side groups, thereby disrupting the normal R-helical conformation. 5166

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Fa Wb %c F W % F W %

It was also concluded that an important condition for the appearance of the band at 1665 cm-1, and hence the RII helix, is the existence of interactions between monomers of BR Langmuir 2009, 25(9), 5159–5167

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forming trimers.37 These R-helical segments are oriented perpendicularly to the membrane plane in either dry or hydrated films.38 Thus, the significant quantity (∼45%) of the RII-helical form found in our measurements suggests that the BR molecules incorporated into PEFs conserve their trimeric organization within the PM patches. During buildup of PEF/PM matrix, vibrations of the methyl branched fatty acid chains originating in phospholipids within the PM were also observed (see Supporting Information).38 To summarize, our FTIR results evidence the stability of the structure of BR in adsorbed form and when incorporated within a PSS/PAH-type PE multilayer.

Conclusion OWLS proved to be well suited to monitor in situ and in real time the buildup of a combined PE/membrane protein matrix: BR in its membrane bound form (PM) was adsorbed and successfully incorporated into the PE multilayered films. The PMs form a single or a double layer when adsorbed on a negatively or positively charged surface, respectively. The formation of thick layers of globular proteins extending to several times the greatest dimension of the protein has already been demonstrated, but this is the first time that such observation is made for proteolipid membranes. The driving forces of the adsorption process of the PM onto charged surfaces were evaluated by varying the ionic strength of the solution as well as the membrane concentration. At high membrane concentration, rearrangement of PE loops cannot take place, presumably due to the kinetic competition by PM adsorption, and the remaining PE protrusions may promote adsorption of a second membrane layer on the positively charged PEF. This cannot be achieved on a negatively charged PEF, or at lower membrane concentration, where PE rearrangement may precede the encounter of further membrane patches from the solution with the surface. PMs were successfully incorporated into the PE multilayers in a way that indicates that they are oriented on the top of the PEFs: when adsorbed on a positive or a (37) Torres, J.; Sepulcre, F.; Padros, E. Biochemistry 1995, 34, 16230–16326. (38) Jonas, R.; Koutalos, Y.; Ebrey, T. G. Photochem. Photobiol. 1990, 50, 1163–117.

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negative surface, they expose the extracellular or cytoplasmic side toward the bulk, respectively. The AFM images revealed the morphology of the PMs adsorbed on either the positively or the negatively charged PE surface. Minor structural changes of BR while adsorbed onto and incorporated within charged surfaces were observed by FTIR spectroscopy in ATR mode. The adsorption does not disturb the secondary structure majority of R-helix and disordered structures of the protein. The R-helix amount remains the same when new PE is adsorbed on the top of the BR layer, demonstrating the very stable structure of this protein in the PM, contrary to earlier observations on adsorbed globular proteins, where β-sheet structures were predominant. Summarizing, charge-driven adsorption of PMs resulted in the formation of highly oriented layers facing the cytoplasmic or extracellular side toward the bulk. Surface charge, ionic strength, and protein concentration are parameters that can be used to produce PE/membrane protein architectures with tunable thickness and hydration properties with a high local organization. Manipulating PMs in this way might promote the development of interesting biomimetic materials for biophotonic, bioelectronic, or sensory devices. Acknowledgment. This work was supported by the Phoremost European Network of Excellence, Project No. 511616: “NanoPhotonics to Realise Molecular Scale Technologies” and by the COST-EU Action MP0702: “Towards functional sub-wavelength photonic structures”. L.Z. is thankful for the grant provided by the Languedoc-Roussillon Region and for the Hungarian Scientific Research Fund (OTKA T049207). Supporting Information Available: The measured refractive indices of the solutions used in this study as a function of ionic strength and protein concentration; the surface topography and profilometric sections of the PMs adsorbed on the top of the multilayered PEFs; the FTIR spectrum of the last five layers of the PEF/PM structure in the range of 28003150 cm-1. This material is available free of charge via the Internet at http://pubs.acs.org.

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