Probing Protein−Membrane Interactions Using Solid Supported

ARTICLE pubs.acs.org/Langmuir

Probing Protein-Membrane Interactions Using Solid Supported Membranes Ann Junghans,† Chlo
e Champagne,†,‡ Philippe Cayot,‡ Camille Loupiac,‡ and Ingo K€oper†,§,* †

Max Planck Institute for Polymer Research, Mainz, Germany Equipe EMMA, Universit
e de Bourgogne, Dijon, France § School of Chemical and Physical Sciences, Flinders University of South Australia, Adelaide, Australia ‡

bS Supporting Information ABSTRACT: Tethered bilayer lipid membranes have been used as a model system to mimic the interactions between the whey protein βlactoglobulin and a lipid interface. The approach allowed for a detailed study of the lipid-protein interactions, the results being of possible importance in food and cosmetic applications. For such applications, lipid-protein interactions and the interfacial behavior are vital factors in controlling and manipulating process conditions such as emulsion stabilization and gelification. Lipid composition as well as the structural properties of the protein governed their interactions, which were probed by a combination of surface plasmon spectroscopy, neutron reflectivity, and electrochemical impedance spectroscopy. Comparison of results obtained using native and a partially unfolded protein indicated that the protein preferentially forms loosely packed layers at the lipid interface.

’ INTRODUCTION Proteins constitute a significant group of natural emulsifiers, and many of their functional properties derive from their structure.1,2 The conformation of proteins at a defined interface has a significant influence on the properties of that interface.3-5 For example, the protein conformation determines the stability and the formation of foams (air-water interface) or emulsions (oil-water interfaces).6 Thus, a detailed knowledge of the interfacial structure of adsorbed emulsifiers such as proteins and lipids on a micro- and nanometer lengths scale and their interfacial properties will have an important role for innovations in drug delivery, cosmetic and food dispersion formulation, for example emulsions and foams. In many applications, surface pressure or surface density, surface composition or film formation determine final product properties.7 However, the underlying processes at a molecular scale are often complex and too difficult to study systematically. Therefore, model systems such as the model protein β-lactoglobulin (βlg) can be used. βlg is one of the major components of the whey proteins from bovine milk and belongs to the lipocalin family. The globular protein is easily isolated from raw milk,8 and has been used in the past as model protein in investigations at the air/water or oil/water interface.9,10 Whey proteins are wellknown in food formulation for their abilities to stabilize emulsions,11 and foams,12 to form gels,13 as well as for their influence on aroma perception.14 r 2011 American Chemical Society

βlg is a small globular protein with 162 residues and a molecular weight of 18.4 kDa. The compact core of the protein consists of a short R-helix segment and eight strands of antiparallel β sheets. βlg has a hydrophobic binding site in its interior, and a weaker one on its exterior, which can bind to various hydrophobic ligands, mainly retinol and long-chain fatty acids.15-17 At room temperature, neutral pH and physiological conditions the native protein exists as a dimer,18 while at concentration below 2 mg/mL the monomeric form is predominant.19 In raw milk, proteins and phospholipids coexists with milk fat globule membranes.20 This membrane is a trilayer of about 10-50 nm thickness, with an electron dense material on the inner membrane face composed of proteins and polar lipids, and a bilayer membrane which consist of 27% (w/w) phospholipids.21 Therefore, studies of proteins at the interface of phospholipids can give important information about processes occurring during protein-fat contacts.22-24 The detailed interaction processes are complex and yet not fully understood.25 In general, during the homogenization of fat, proteins adsorb at interfaces due to hydrophobic and hydrophilic interactions. During these adsorption processes, the proteins typically unfold partially or denature. The extent of unfolding of globular proteins depends on various factors such as surface pressure, surface Received: August 11, 2010 Revised: January 6, 2011 Published: February 14, 2011 2709

dx.doi.org/10.1021/la103200k | Langmuir 2011, 27, 2709–2716

Langmuir potential, and hydrophobicity of the surface. The fat homogenization process destroys native fat globules (3-7 μm of diameter) and produces new fat globules (0.3-0.7 μm of diameter) stabilized essentially by milk proteins. Phospholipids originally present in raw fat globules can be neglected. In order to facilitate the systematic investigation of such interaction processes, model interfaces can be used. Lipid bilayers formed of triglicerides and cholesterol, and the protein βlg constitute not only a model to understand protein denaturation during milk fat homogenization but also a model to study the proteins in a bilayer such as in a cell membrane. Here, a solid-supported model membrane system has been utilized.26 Tethered bilayer lipid membranes (tBLMs) consist in principle of a lipid bilayer that is covalently attached to a solid support via an oligomeric spacer group.27 They have been shown to provide excellent electrical sealing properties and have therefore been used in investigations of ion channel proteins and have been proposed for biosensor applications.28-31 Furthermore, tBLMs can serve as model platform to study membrane related processes, such as protein-lipid interactions. The membrane platform is accessible to a wide variety of surface analytical tools, which allows studying for example the adsorption of a protein on a membrane in detail. Here, surface plasmon resonance spectroscopy (SPR), neutron reflectivity (NR) and electrical impedance spectroscopy (EIS) have been used to investigate different aspects of the membrane-protein system. By using SPR spectroscopy, changes in the local refractive index at an interface are detected, thus giving information about the ordering of the layers on a macroscopic scale. The scattering length density (sld) profile obtained by NR measurements probes the surface architecture on a smaller length scale. Finally, the electrical properties of the system were measured by EIS, giving indication about possible penetration of the protein into the lipid layer. The interaction of native and denatured βlg with membranes of different composition was probed. The native protein was only able to penetrate the lipid bilayer, if the membrane was not very densely packed. The partially unfolded protein, on the other hand, formed a dense layer at the membrane interface.

’ MATERIALS AND METHODS Chemicals. Ultra pure water, filtered with a Millipore device (Billerica, MA) was used throughout the experiments. SPR and EIS measurements were performed in either 0.1 M NaCl solution (Sigma-Aldrich, Munich, Germany g99.5%) or in PBS buffer (0.1 M, pH 7) prepared using Na2HPO4 and NaH2PO4 (both Fluka, Munich, Germany g99%). Lipids. The anchor lipid DPhyTL (2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-D,L-lipoic acid ester lipid) was synthesized as described before.32 DPhyPC (Diphytanyl phosphatidylcholine) was obtained from Avanti Polar Lipids (Alabaster, AL). Triolein (Glyceryl trioleate, practical grade, ∼65% purified) and Cholesterol (Sigma grade, g99%) were purchased from Sigma-Aldrich (Munich, Germany). Protein Purification. β-Lactoglobulin was purified from whey protein powder (Bipro, Davisco, Geneva, Switzerland). The powder was slowly dissolved in water (73.5 g of whey powder/L) under constant agitation for at least 2 h (preferably overnight). Trichloroacetic acid (Serva, Heidelberg, Germany) was used as a precipitant (3.1 g in 20 mL of H2O per 100 mL of protein solution) solution. The mixture was centrifuged (10000 g, 30 min, 20 °C) and the supernatant was dialyzed (cutoff 3500

ARTICLE

Da) under constant agitation 3 times for 2 h against water and 3 times for 2 h against PBS. Sodium azide (0.2 g/L) (Acros, Geel, Belgium) was added to avoid microbial degradation of the sample. The concentration of the protein was probed by UVvis spectroscopy (Lambda900, Perkin-Elmer, Waltham, MA). Absorbance of the diluted protein was measured in the range of 350-200 nm. The protein concentration was determined using the absorbance intensity at 278 nm and ε(0.1%) = 0.96. The concentration was about 20 g/L, with slight variations for every sample batch, due to small variations in dialysis time and amount of material in the dialysis cell. SDS page electrophoresis was used to verify the purity of the protein after purification. A microcalorimeter DSC III (Setaram Instrumentation, France) has been used in order to determine denaturation temperature (Td) and enthalpy (ΔH) of protein unfolding for βlg in solution. A temperature gradient of 0.5 °C/min was used between 25 and 110 °C. About 600 mg of protein solution (15 g/ L of protein) was placed in a hermetically closed cell. PBS buffer solution (0.1 M) was used as a reference. From the obtained thermograms, Td and ΔH were calculated by integration of the measured curves. For H/D exchanged protein preparations, the last three dialysis steps were carried out against PBS prepared with D2O (Aldrich, Munich, Germany 99.9%). For the experiments with denatured β-lactoglobulin, the protein was unfolded by urea denaturation.33 After the normal purification procedure, the supernatant was first dialyzed for 2 h against PBS with 6 M urea, and finally against normal PBS, as described above. Solid substrates. For SPR measurements, a high refractive index glass (LaSFN9, Hellma Optik, Jena, Germany) was coated with a thin chromium layer (2.5 ( 0.5 nm) followed by 50 ( 0.5 nm of gold. For NR investigations, silica wafer were coated with 2.5 ( 0.5 nm of chromium and 20 ( 0.5 nm of gold. EIS measurements were conducted on template stripped gold slides.34 Membrane Assembly. DPhyTL monolayers were self-assembled on the gold films by immersion of the substrate in a lipid solution (0.2 mg/mL in ethanol) for 24 h. Before use, samples were cleaned with ethanol (Sigma-Aldrich, Munich, Germany) and dried with nitrogen. Monolayers were completed to bilayers by fusion with small unilamellar vesicles (50 nm by extrusion, 2 mg lipid per mL of H2O) or by rapid solvent exchange (RSE). Here, the substrate is incubated in an ethanolic lipid solution of 5-10 mg lipid in ethanol, depending on the used lipid. After 10 min, the cell is rapidly, and in large quantity (approximately 20
cell volume), flushed with aqueous buffer. During this exchange, the water insoluble lipid molecules complete the bilayer on top of the phytanoyl chains of the monolayer, rather than to mix with the water and get rinsed away. This preparation technique is suitable for forming defect-free bilayers over large substrates as have been used for neutron reflectivity measurements. For RSE, 5 mg/mL DPhyPC and 10 mg/mL glyceryl trioleate in ethanol were used. For mixed layers, 10 w% cholesterol was mixed with either DPhyPC or triolein and dissolved in ethanol. The membranes were formed by RSE as described above. Surface Plasmon Resonance Spectroscopy. A customized SPR setup in the Kretschmann configuration with a 632 nm He/ Ne laser was used. In the scan mode, changes in the reflected intensity are measured as a function of the scattering angle, while in the kinetic mode, changes at a fixed angle are detected as a function of time. Measurements were performed in a Teflon flow 2710

dx.doi.org/10.1021/la103200k |Langmuir 2011, 27, 2709–2716

Langmuir

ARTICLE

Table 1. Optical Parameters Used To Fit the SPR Dataa compound

ε real

ε imaginary

DPhyTL

2.2534

0

DPhyPC

2.102534

0

triolein

2.035

1.8

βlg

2.036

0

a

Due to the inhomogeneous nature of the triolein layer, an imaginary part had to be introduced, taking into account adsorption effects.

Figure 2. Scattering lengths density profiles for the adsorption of βlg on a DPhyTL monolayer measured in D2O and a mixture of D2O and H2O. For the scattering data, see Figure S1 in the upporting information.

Figure 1. SPR adsorption kinetics of βlg on DPhyTL monolayers and DPhyTL/DPhyPC bilayers. Native and partially unfolded protein was added and the increase in reflectivity was measured at a fixed angle of incidence. Oscillations in the signal are due to fluctuations in the laser intensity.

cell. Data was evaluated using Fresnel equations. The system was analyzed in terms of a box model using the parameters listed in Table 1. Electrical Impedance Spectroscopy. EIS was performed using an Autolab PGSTAT 12 (Eco Chemie, Utrecht, Netherlands). Spectra were recorded for frequencies between 2 mHz and 100 kHz at 0 V bias potential with an AC modulation amplitude of 10 mV. Experiments were performed in a Teflon flow cell with a buffer volume of 1 mL and an electrochemically active area on the substrates of about 0.28 cm2. Three electrode measurements were performed with the substrate as the working electrode, a coiled platinum wire as the counter electrode, and a DRIREF-2 reference electrode (World Precision Instruments, Berlin, Germany). Data were analyzed using an R(RC)C-circuit (cf. Figure S3, Supporting Information),27,34 where the bilayer is represented by a parallel circuit of a resistor and a capacitance. This RC element is in series with a capacitance, representing the region below the membrane with the spacer molecules and the gold surface. A final resistor corresponds to the influence of the electrolyte solution. Neutron Reflectometry. All neutron reflectivity investigations were carried out at the NG1 or AndR beamline at the NIST Center for Neutron Research, Gaithersburg, MD. The different assembly steps of the system (monolayer, bilayer, protein) were followed sequentially by performing NR measurements in two different contrasts, namely in D2O and in an H2O/D2O mixture (2/3D2O þ 1/3H2O) with a scattering length density (sld) of about 4
10-6 Å-2 (CM4). Using solutions of different sld results in complementary data sets, which subsequently can be fitted simultaneously.

Analysis of the NR data was conducted in three steps. Data reduction was performed using reflred. The data was then analyzed using reflfit and finally using GaRefl. All programs were developed by P.A. Kienzle et al. and can be found on the Web site of the NIST Center for Neutron Research. A detailed description of the fitting procedure has been published recently.37 Fluorescence Spectroscopy. The measurements were performed on a spectrofluorimeter from Perkin-Elmer (LS-45/55) in a 1 cm quartz cell at 25 °C, with the excitation and emission slits at 2 nm. The protein concentration for these experiments was about 0.4 g/L (2
10-5 mol/L), potassium phosphate buffer at 0.1 M and pH 7 was used. For tryptophan fluorescence measurements, the excitation wavelength was 291 nm and emission spectra were recorded in the wavelength region from 300 to 450 nm.

’ RESULTS AND DISCUSSION In order to systematically study the interaction of βlg with lipid layers, various membrane architectures have been prepared and interfaced with protein solutions. Protein on DPhyTL. First, the protein has been adsorbed onto hydrophobic DPhyTL monolayers. The SPR experiments showed an adsorption process over about 40 min (Figure 1). Assuming a refractive index for the protein of n = 1.4136 and an exclusive adsorption of the protein on top of the monolayer, the increase in reflectivity corresponded to a layer thickness of about 30 Å. This would correspond to a complete layer of flattened protein.25,38 However, this model assumes that the protein would not penetrate the hydrophobic part of the DPhyTL layer. In order to further investigate this behavior, NR experiments were performed (Figure 2). NR is an ideal tool to study buried interfaces, and should give indications, whether the protein actually penetrated the monolayer. The experimental data showed small changes in the intensity at smaller Q after the addition of βlg to a DPhyTL monolayer. However, the fitting of the data allowed for two different models. In the first approach, the adsorption of the protein led to a layer on top of the monolayer with a thickness of 26 Å, results in agreement with those obtained by SPR. Yet, the data could also be fitted to a model allowing for a slight penetration of the protein into the hydrophobic part, leading to changes in the slds of the hydrophobic region and the spacer part and a thin additional protein 2711

dx.doi.org/10.1021/la103200k |Langmuir 2011, 27, 2709–2716

Langmuir

ARTICLE

Figure 3. (A) DSC thermograms for native, denatured and partially refolded protein. (B) Corresponding fluorescence spectra.

layer of about 9 Å. Comparison of SPR and NR data lets us favor the first model. The layer thickness suggests that the protein remains in it native state or only partially unfolds.25 It could have been suggested that the interactions with the hydrophobic layer (water contact angles of about 110°) would lead to a more pronounced unfolding of the protein, and a thicker layer of aggregated proteins. Indeed, lipids are typically good solvents for the hydrophobic regions of a protein, an oil phase will effectively solvate the hydrophobic amino acid residues. In the native form, some helical regions and the β-sheet hydrophobic core of βlg are buried in the interior of the protein, which can only bind the lipids if the protein is denatured and unfolded. Interaction with Denatured Protein. In order to further investigate the possibility of protein denaturation at the lipid interface, experiments with predenatured protein have been performed. Temperature can induce substantial changes to the protein conformation.39,40 Heat denaturation can cause aggregation of proteins, e.g., due to the formation of intermolecular disulfide bridges. In order to exclude such effects, the protein was (partially) unfolded by urea.33 The level of unfolding and refolding of the protein after different denaturation steps was investigated by DSC and intrinsic (tryptophan) fluorescence spectroscopy (Figure 3). The thermogram of the native protein showed a typical endothermic peak at around 68 °C with a denaturation enthalpy of about 18 mJ/g protein. After dialysis against 6 M urea in potassium phosphate (Kpi) buffer, the endothermic peak disappeared. The apparition of an exothermic peak indicated aggregation of unfolded proteins, yet only at higher temperatures. The protein could be partially refolded by dialysis against phosphate buffer overnight. The denaturation enthalpy of the refolded protein was about 10 mJ/g protein, indicating an only partially refolded protein structure. The effect of denaturation on the structure of the protein has been further investigated by tryptophan fluorescence (Figure 3B). βlg has two tryptophan residues; one is situated at the surface, the other one in the core of the protein. The native protein gave thus an average signal for both tryptophan units, resulting in a maximum in intensity at 325 nm. After the urea treatment, the intensity increased and a red-shift could be observed, indicating that the tryptophan moieties are exposed to a more polar surroundings. During the denaturation process, the protein unfolds and the inner core region becomes exposed. After refolding, the fluorescence emission shifts toward the initial values, yet reaching neither the native wavelength nor intensity.

This supports the DSC observation that the protein only partially refolds back to the initial structure, obtaining a less compact form. When this partially unfolded protein was added to the hydrophobic DPhyTL monolayer, an increase in reflectivity could be observed. If fitted with the same refractive index as the native protein, this would correspond to a protein layer of about 50 Å (Figure 1). Yet, a more unfolded protein would probably have a lower refractive index and thus the effective layer thickness would be higher. Thus, the partially unfolded proteins assemble most probably at the interface to form a loosely packed multilayer. Protein on DPhyTL and DPhyPC. After the experiments probing the interactions with a hydrophobic interface, the protein has been interfaced with lipid bilayers. DPhyTL monolayers were completed by fusion with small unilamellar DPhyPC vesicles to dense bilayers.41 These membranes present a very dense and insulating layer, which typically is only penetrated by hydrophobic molecules such as membrane proteins or ionophores. The addition of βlg to these layers led to no detectable changes in the SPR signal (Figure 1). This indicates that the interactions between the hydrophobic headgroups of the lipids and the protein are not strong enough to either attract the protein to the interface or to allow for a penetration inside the membrane. This assumption is supported by the fact that the addition of the predenatured protein to a DPhyPC bilayer led to a significant increase in reflectivity, corresponding to an additional layer of about 53 Å (Figure 1). In contrast to the experiments performed on the monolayer, there was a slight difference in the results obtained from SPR and NR experiments on the interactions of the native protein and the DPhyTL/DPhyPC bilayer. The scattering length density profile obtained from the NR experiments showed a thin protein layer on top of the bilayer (Figure 4). If fitted with a model where the protein was allowed (but not forced) to penetrate the bilayer, a layer with a thickness of 2 Å was formed on top of the DPhyPC leaflet. Additionally the sld of the spacer part increased from 0.54
10-6 Å-2 to 1.24
10-6 Å-2. Although this increase in the sld of the spacer part of the molecule suggested that the protein entered the bilayer and moved below, this is not very likely as especially DPhyTL forms very dense inner and outer bilayer.41,42 It is more probable that this result is a residue of the fitting procedure as the changes in the neutron reflectivity profiles are very subtle and the spacer inner and outer alkyl chain layers are strongly interconnected and hard to distinguish. The slight interactions with the bilayer observed by NR might be due to the bilayer formation process. For NR experiments, a large 3-in. 2712

dx.doi.org/10.1021/la103200k |Langmuir 2011, 27, 2709–2716

Langmuir

ARTICLE

Figure 4. Scattering lengths density profiles for the adsorption of βlg on a DPhyTL/DPhyPC bilayer measured in D2O and a mixture of D2O and H2O. For the scattering data, see Figure S2 in the Supporting Information.

Figure 5. SPR adsorption kinetics for the addition of native lg to different membrane architectures.

Si wafer was used as substrate, leading to a membrane area of almost 50 cm2. For such a large area, vesicle fusion did not lead to a complete bilayer coverage. However, rapid solvent exchange could be used. This probably led to small defects in the membrane architecture, allowing then for interactions of the protein with the hydrophobic core of the membrane. Similar to results obtained for the monolayer architectures, there was also a significant difference in the SPR response for the interaction of native and predenatured protein with lipid bilayers. The addition of the partially unfolded protein led to an increase in reflectivity corresponding to a layer thickness of about 53 Å, when assuming a similar refractive index (Figure 1). This value is very similar to the one obtained for the partially denatured protein at the hydrophobic interface, suggesting a similar scenario as described above. However, the interactions process appeared to be slowed down in the bilayer case. The reflectivity reached the final intensity only gradually, while a rapid increase had been observed for the adsorption on the monolayer. Influence of the Membrane Density. In order to further elucidate the effect of membrane defects or more general of the lipid composition and overall membrane structure, bilayers with different distal leaflets were formed, still using DPhyTL as the inner leaflet. Triolein is a long-chain monounsaturated fat and triglyceride and the major fat in milk.20 The lipid is a basic food component and an energy storage molecule; e.g., it is the principal component of olive oil, and has considerable importance for food and fuel industries.43 Additionally, triacylglyceride are water-insoluble neutral lipids that serve as the main storage form of fatty acids in most animals and plants. Using RSE techniques, a triolein layer could be deposited on a DPhyTL monolayer. For the analysis of the SPR data, a theoretical layer thickness of a triolein layer of 30 Å was used. The theoretical dielectric constant ε0 of triolein is 2.15, however during experiments typically values of about 2.0 or below have been found. Additionally, an imaginary part ε00 = 1.8 had to be introduced to account for internal scattering in the distal bilayer leaflet. This indicates that the layer probably was not complete, or had a significant amount of defects. The addition of βlg to this bilayer led to a pronounced increase in the SPR reflectivity, corresponding to a layer thickness of about 20 Å (Figure 5). This would support the assumption that the protein favorably interacts with the hydrophobic part of the membrane. A triolein layer

with a relatively high defect density would expose more hydrophobic part than for example a dense DPhyPC bilayer, were no interaction was visible. Additionally, when a DPhyPC layer was formed in the SPR cell using RSE techniques, the protein addition also led to an increase in reflectivity, similar to the results observed by neutron reflectivity. In comparison to the very dense layer formed by vesicle fusion,41 the RSE bilayer will have more defects and thus expose more hydrophobic sites for protein interaction. This hypothesis has been further investigated by adding cholesterol into the distal membrane leaflet. Cholesterol is known to enhance membrane fluidity, by intercalating between the lipids. It can thus seal eventual defects in the membrane architecture. In the case of a triolein layer with 10% cholesterol, a decrease in adsorption of βlg compared to a pure triolein layer could be observed (Figure 5). The protein only formed a layer of about 13 Å. The cholesterol molecules probably integrated very well into the loose triolein layer, filling the defects and rendering the membrane denser. The protein was thus interfacing a more hydrophilic layer and had fewer possibilities to interact, unfold, aggregate, or adsorb. DPhyPC already formed an almost perfect bilayer. Compared to the triolein layer, the addition of cholesterol to DPhyPC had an opposite effect. The mixed layers were apparently less ordered, leading to an increased interaction with the added protein. Yet, the interaction was still less than between the protein and the cholesterol/triolein layer. These observations are in line with previous experiments performed at the air/water interface using lipid films of various lipid density. Electrochemical Impedance Measurements. The interaction between βlg and the different membrane assemblies was further investigated using electrochemical impedance spectroscopy. While SPR gives optical information about the formation of different layers at the interface, EIS can probe the electrical properties of the membrane in terms of capacitive and resistive properties. Thus, not only the addition of layers to the membrane can be observed, but also the possible integration of the protein inside the membrane would be detectable. A lipid bilayer typically can be described electrically as a combination of a resistor (R) and a capacitance (C). The values of the RC elements can be obtained, by fitting the EIS data to an equivalent circuit.27,44 Especially the capacitive values can give valuable information about the processes occurring at the membrane 2713

dx.doi.org/10.1021/la103200k |Langmuir 2011, 27, 2709–2716

Langmuir

ARTICLE

Figure 6. Frequency reduced admittance plots describing the interaction of βlg on different membrane architectures. Black symbols represent the bilayer, while gray symbols are measured after protein additions. Solid lines represent fitting of the data using an R(RC)C equivalent circuit. The insets depict schematically the membrane-protein interactions. The membranes were all build on DPhyTL monolayers with the following distal layers: (A) DPhyPC, prepared by RSE, (B) triolein, (C) DPhyPC with 10% cholesterol, (D) triolein with 10% cholesterol, (E) DPhyPC, prepared from vesicles, and (F) triolein. For parts A-D, a native form of the protein was added while for parts E and F a partially unfolded protein was used.

interface. For example, for a perfect RC element, the capacitance is inversely proportional to the thickness of the dielectric layer. An increase in layer thickness, e.g., the formation of a protein layer on top of the membrane, would thus lead to a decrease in the membrane capacitance. On the other hand, the incorporation of the protein inside the membrane would change the dielectric properties of the latter and would lead to an increase in capacitance. The different membrane architectures, i.e., DPhyPC and triolein layer with and without cholesterol and their interaction with the protein were probed using EIS (Figure 6). The different membrane compositions have an effect on the respective electrical properties. In order to compare the effect of the protein on the membrane, relative changes were compared. The changes in capacitance can be clearly seen in an admittance plot, where the RC element that describes the bilayer properties can be observed as a semicircle.34 (An example for a Bode plot representation can

be seen in Figure S3 in the Supporting Information.) The intercept with the y-axis corresponds to the magnitude of the capacitance. On a DPhyPC bilayer formed by vesicle fusion, no change could be detected after addition of the protein, in good agreement with SPR results (see Supporting Information). However, the protein had an effect on the electrical properties of the other membranes. Similar to the SPR results, the effects on membranes formed by RSE using DPhyPC or DPhyPC with 10% cholesterol were very small. Namely, the capacitance values changed by less than 1% and about 1%, respectively. These results underline that the interactions between βlg and dense DPhyPC membranes are weak, the proteins interact only slightly with the lipid layer, the increase in capacitance suggest a slight penetration if cholesterol is added to the distal membrane leaflet. In the case of triolein layers, the addition of βlg led to a pronounced increase of the membrane capacitance. Again, similar to the SPR data, a pure triolein layer showed the highest 2714

dx.doi.org/10.1021/la103200k |Langmuir 2011, 27, 2709–2716

Langmuir

ARTICLE

Table 2. Summary of the SPR, NR, and EIS Resulta protein conformation

SPR layer thickness/Å

NR layer thickness/Å

% change in capacitance

DPhyTL

native

30 ( 2

26

n/a

DPhyTL

urea denatured

50 ( 2

n/a

n/a

DPhyTL þ DPhyPC (prepared by RSE)

native

DPhyTL þ DPhyPC (prepared from vesicles)

urea denatured

0(2

DPhyTL þ DPhyPC/Cholesterol

native

9(3

n/a

þ1

DPhyTL þ triolein

native

20 ( 3

n/a

þ 10

53 ( 2

2