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Langmuir 1996, 12, 5636-5642
Formation and Characterization of Lipopeptide Layers at Interfaces for the Molecular Recognition of Antibodies† Mila Boncheva,‡ Claus Duschl,‡ Werner Beck,§ Gu¨nter Jung,§ and Horst Vogel*,‡ Institut de Chimie Physique, De´ partement de Chimie, Ecole Polytechnique Fe´ de´ rale de Lausanne, CH-1015 Lausanne, Switzerland, and Institut fu¨ r Organische Chemie der Universita¨ t Tu¨ bingen, Auf der Morgenstelle, D-72076 Tu¨ bingen, Germany Received June 11, 1996. In Final Form: August 26, 1996X A lipopeptide, carrying the antigenic peptide segment 135-154 of VP1, one of the capsid proteins of the picornavirus which causes foot-and-mouth disease in cattle, was investigated in lipid monolayers on the water surface and on hydrophobic solid supports. Such lipid monolayers, if present in the fluid state, can incorporate the lipopeptide at any given lipid/lipopeptide ratio. Circular dichroism and infrared spectroscopy show that, on the surface of the lipid layers, the peptidic portion of the lipopeptide adopts an average structure similar to that observed on the surface of the intact virus. The peptide is fully accessible to specific antibody binding as revealed by epifluorescence microscopy of lipid monolayers on the water surface and by surface plasmon resonance measurements of supported layers. In addition, the lipid monolayer surface prevents nonspecific protein binding. Since the lipopeptide-containing lipid monolayers can easily be formed by self-assembly on many transducer surfaces, we believe that this method is of general importance for the controlled presentation of antigens for the subsequent detection of antibody binding. More importantly, the findings open the way for simulating ligand-receptor interactions on biological cell surfaces in a reconstituted system using modern surface sensitive techniques which are equally interesting for basic research and biosensor applications.
Introduction The binding of ligands to cell surface receptors is fundamental for biological signal recognition and amplification reactions. The elucidation of these events is one of the most important issues in modern biological research. Elementary signal transduction mechanisms not only are of interest for basic research but also have become increasingly attractive for the construction of a new generation of biosensors. The first step toward the realization of such a goal is the development of suitable methods for immobilization on the surface of sensor devices the particular membrane components necessary for signal detection and amplification in a functionally active form. To this end, the covalent attachment of phospholipid membranes on gold and glasslike surfaces for the formation of robust and stable bilayers suitable for the reconstitution of transmembrane receptor proteins has been intensely studied in the last years.1-3 The present work concerns the specific interaction of lipid-anchored antigenic peptides with antibodies at the surface of supported lipid layers. Two major issues are of interest in this context. Firstly, to investigate the formation of lipid-attached peptide layers as a general route for the presentation of functional active peptides on sensor surfaces. Secondly, to show the feasibility of this approach in the field of biosensors applicable in immunological research and vaccine development. For this purpose we have chosen the lipopeptide (LP) shown schematically on Figure 1 as a model compound. It carries the peptide sequence 135-154 of VP1, one of * To whom correspondence may be addressed. † This work was performed by M.B. at the EPFL as a part of her Ph.D. thesis. The lipopeptides were synthesized at the University of Tu¨bingen by W.B. ‡ Ecole Polytechnique Fe ´ de´rale de Lausanne. § Institut fu ¨ r Organische Chemie der Universita¨t Tu¨bingen. X Abstract published in Advance ACS Abstracts, October 15, 1996. (1) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197-210. (2) Heyse, S.; Vogel, H.; Sa¨nger, M.; Sigrist, H. Protein Sci. 1995, 4, 2532-2544. (3) Naumann, R.; Jonczyk, A.; Kopp, R.; van Esch, J.; Ringsdorf, H.; Knoll, W.; Graeber, P. Angew. Chem., Int. Ed. Engl. 1995, 34, 20562058.
S0743-7463(96)00575-6 CCC: $12.00
the capsid proteins of the picornavirus causing foot-andmouth disease in cattle (for reviews, see refs 4 and 5). This segment has been shown to represent one of the major antigenic sites of the virus. Its three-dimensional structure has been solved by X-ray diffraction.6 In the present study the peptide was chemically attached to the lipopeptide Pam3CysSerSer, a chemical analogue of the lipid anchor of membrane-bound lipoprotein in Escherichia coli.7 Since lipopeptides designed according to this principle exhibit excellent carrier and adjuvant properties for antibody production, they have also been used in the development of synthetic, low-molecular-weight vaccines.7 Biosensors based on immobilized lipopeptides might be well-suited for screening antibody production during vaccine development, as well as for detecting the presence of antibodies in the blood in order to discover viral infection at an early stage. The amphiphilic nature of the LP molecule offers a simple way for its vectorial assembly in monomolecular layers at the air/water interface, on hydrophobized solid substrates, and in bilayers, either in pure form or mixed with phospholipid molecules. The experimental section of this work can be structured in three parts. In the first part we investigated the conformation of the peptide moiety of the LP in bilayers of either pure LP or in LP/ phospholipid mixtures and compared it with the known X-ray structure of the corresponding peptide segment in the intact virus. Here we assume that the conformation of the peptide moiety in lipid bilayers resembles that found in lipid monolayers reasonably well. To investigate the LP secondary structure, we used two well established complementary spectroscopic methods: circular dichroism (CD) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. While the helical content of the polypeptide secondary structure can be determined reasonably well by CD experiments, the (4) Belsham, G. J. Prog. Biophys. Mol. Biol. 1993, 60, 241-260. (5) Becker, Y. Virus Genes 1994, 8, 199-214. (6) Logan, D.; Abu-Ghazaleh, R.; Blakemore, W.; Curry, S.; Jackson, T.; King, A.; Lea, S.; Lewis, R.; Newman, J.; Parry, N.; Rowlands, D.; Stuart, D.; Fry, E. Nature 1993, 362, 566-568. (7) Metzger, J.; Wiesmu¨ller, K.-H.; Schaude, R.; Bessler, W. G.; Jung, G. Int. J. Pept. Protein Res. 1991, 37, 46-57 and references therein.
© 1996 American Chemical Society
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Langmuir, Vol. 12, No. 23, 1996 5637
Figure 1. (A) Correlation between the structure of the lipopeptide and the coat protein of the virus. Shown schematically are the three-dimensional structure of the VP1 virus protein with the βG-βH loop indicated, a magnified view of the loop structure (amino acid sequence 134-154), and finally the lipopeptide structure with the peptide part represented by a sequence of black balls (from ref 6, with modifications). (B) Chemical structure of the lipopeptide. The VP1 polypeptide 134-154 (indicated in the frame) is covalently linked to the lipotripeptide Pam3CysSerSer. The charged amino acid residues in the polypeptide sequence are indicated. The three palmitoyl chains are represented by zigzag lines.
measurements are less reliable with respect to β-structures and β-turns.8 On the other hand, the contributions of water, R-helical and nonregular structures in the Amide I region of the infrared spectrum are often difficult to distinguish, but the method is quite confident about the content of other secondary structures. In the second part of the work, the molecular dimensions and the phase behavior of pure LP and mixed LP/lipid monolayers were studied by Langmuir film balance techniques. Furthermore, the binding of antibodies from the bulk aqueous phase to the peptide moiety of the LP monolayer at the air/water interface was directly observed by epifluorescence microscopy using fluorescently-labeled molecules (antibodies and/or LP). The third part of this work concerns the investigation of LP in supported monolayers on thioalkylated gold surfaces by surface plasmon resonance (SPR). Because this technique offers a sensitive way of measuring mass coverage at metal/dielectric interfaces, it was possible to quantify the antigen content in the supported layer and the binding of antibodies to it. Materials and Methods The lipopeptide Pam3Cys-Ser-Ser-[VP1(135-154)] (molecular mass 3417) and its NBD-labeled analogue (NBD-LP) were synthesized as described elsewhere.7 The Pam3Cys was purchased from Boehringer-Mannheim. These compounds were stored as dry powders at -50 °C and dissolved prior to use to give stock solutions of 1 mg/mL in methanol. The solutions were stored at 4 °C. The monoclonal antibodies (Ab) against the VP1(135-154) were produced by Dr. J. Bouvier, Ciba Geigy, St. Aubin, Switzerland. The exact LP and Ab concentrations were determined by the UV absorbance at 280 nm using molar extinction coefficients of 1100 and 227 000 M-1 cm-1, respectively. The tetradecanethiol (purity >95%) and the lipids 1-palmitoyl2-oleoylphosphatidylcholine (POPC), 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), and 1,2-dipalmitoylphosphatidylcholine (DPPC) were from Fluka (Buchs, Switzerland) and used without (8) Vogel, H. Biochemistry 1987, 26, 4562-4572.
further purification (purity >99% as tested by thin layer chromatography) as stock solutions of 1 mg/mL in methanol and 2 mg/mL in chloroform/methanol (9/1), respectively. The water used in all experiments was purified in an ion-exchanger purification train (Milli-Q system, Nanopore, Volketswil, Switzerland) and had resistivity higher than 18 MΩ cm. Unless otherwise indicated, the buffer used was 5 mM phosphate, pH 7.4. The organic solvents and the salts used for buffer preparation were purchased from Fluka (Buchs, Switzerland) and were of the best quality available. The antibodies were fluorescently labeled with Rhodamine-X isothiocyanate (Molecular Probes Inc., Eugene, OR) according to standard procedures9 to a final ratio of about two Rhodamine-X molecules per antibody, as determined by absorption spectroscopy using the ratio of the intrinsic protein absorption at 280 nm to the Rhodamine absorption at 570 nm. Circular Dichroism Measurements. CD experiments were performed on an AVIV model 62 DS circular dichroism spectrometer (AVIV, Lakewood, NJ). Spectra were recorded between 186 and 250 nm (step resolution of 2 nm) using a quartz cuvette (path length 0.1 mm, sample volume 40 µL) at 20 °C. Each experiment was repeated four times. Reference samples of the respective solvent (methanol or buffer containing 1 mM TrisHCl and 1 µM EDTA, pH 7.4), dispersions of lipids or Pam3Cys were routinely recorded and subtracted from the original spectra. Since the dichroic behavior of peptides in the far-UV up to 250 nm is determined predominantly by their secondary structure, the spectra were used to determine the conformation of the LP in the different sample preparations: methanol solution (for the pure LP) and mixed vesicles (for the pure LP and the LP/lipid mixtures). In order to obtain the secondary structure of the LP, its CD spectrum was fitted to a reference set of 15 proteins of known three-dimensional structure using the modified method of Hennesey and Johnson10 as described elsewhere.8 The fit determines the weight of the individual reference proteins in the measured spectrum. Since the three-dimensional structure of the reference proteins is known, it is in turn possible to evaluate (9) Harlow, E.; Lane, D. Antibodies-a laboratory manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1988. (10) Hennesey, J. P.; Johnson, W. C., Jr. Biochemistry 1981, 20, 10851094.
5638 Langmuir, Vol. 12, No. 23, 1996 the mean secondary structure of the lipopeptide in the corresponding preparations as percentages of helical, β-strand, β-turn, and undefined structural motifs. Attenuated Total Reflection Fourier Transform Infrared Spectroscopy. The experiments were performed on a Bomem Model 110 Michelson interferometer (Bomem, Montreal, Canada) with a narrow-band, liquid nitrogen cooled HgCdTe detector. The resolution was 4 cm-1 and triangular apodization was used. The overhead ATR unit (Specac, England) consisted of a horizontally mounted 22.5° trapezoidal Ge crystal. The whole instrument was continuously flushed with nitrogen. The light beam reflected above the critical angle inside the Ge plate creates an evanescent field penetrating a few micrometers into the sample, thus probing the structure of molecules at the Ge surface. A total of 400 scans were typically collected for the sample spectra. Spectra of water vapor and of the bare crystal were routinely recorded and subtracted from the original sample spectra. The sample (typically 50 µg of LP dissolved in chloroform/methanol (9/1), if necessary premixed with lipid in the desired molar ratio) was spread on the ATR crystal. The 1500-1800 cm-1 spectral region of the spectra was analyzed as a sum of Gaussian/ Lorentzian curves in a linear least-squares fit routine, where the amplitudes, band positions, half-widths, and the Gaussian/ Lorentzian composition were optimized consecutively (for details of the fitting routine, see ref 11). The band positions in the Amide I region (1600-1700 cm-1), associated mainly with the stretching vibrations of the carbonyl groups of the peptide bonds, are indicative of the peptide secondary structure. Monolayer Experiments. For the monolayer experiments we used a computer-controlled Langmuir film balance (Riegler & Kirstein, Mainz, Germany). The trough and the barriers were made of Teflon, and the pressure was measured via a Wilhelmy plate. For the fluorescence microscopy experiments, the film balance was mounted on the object table of a Zeiss Axiotron epifluorescence microscope (Zeiss, Germany). Two different filter sets allowed the separate excitation and observation of the NBDlabeled LP in the monolayer and the Rhodamine-X-labeled Ab. In order to reduce thermal convection, the trough and microscope objective were enclosed in a plastic box. The LP and the LP/lipid monolayers were spread on the water surface with a Hamilton microsyringe from chloroform/methanol (9:1) solutions. After the solvent evaporation (10 min), the monolayers were compressed at a speed of 0.05-0.1 nm2 molecule-1 min-1. For the Ab-binding experiments, a known amount of the Ab stock solution was injected into the subphase and stirred with a small magnetic bar placed on the trough bottom. Surface Plasmon Resonance Measurements. The SPR experiments were performed on a home-made setup in Kretschmann configuration as previously described.12 Briefly, linearly p-polarized light with a wavelength of 632.8 nm was directed from a He-Ne laser (Uniphase, 1103p) through a 60° glass prism (SF10, Spindler & Hoyer) to a gold film. Using a photodiode, the reflected light intensity was measured either as a function of angle of incidence (step resolution of 0.05°) or as a function of time at a fixed angle of incidence. The data were recorded using a lock-in amplifier and transferred to a personal computer using software developed in our group. SPR is a surface sensitive technique. Upon total internal reflection of a laser beam on the prism base, surface plasmons can be excited at the metal/dielectric interface. Their coupling to the evanescent wave of the incident light is observed as a minimum in the reflectivity vs angle of incidence curve at the resonance angle θ. The evanescent field of the surface plasmons extends into the dielectric medium and is highly sensitive to its optical properties near the interface. Covering of the metal with an organic layer typically shifts the position of the resonance angle, θ, to higher values. The measurement of ∆θ allows determination of the optical thickness of the adsorbed layer using ∆θ ) k∆nd, where k is a constant reflecting the experimental conditions, d is the geometrical thickness of the layer, and ∆n is the difference between the real refractive indices of the layer and the medium.13 The mass coverage on the gold surface can (11) Thiaudie`re, E.; Soekarjo, M.; Kuchinka, E.; Kuhn, A.; Vogel, H. Biochemistry 1993, 32, 12186-12196. (12) Terrettaz, S.; Stora, T.; Duschl, C.; Vogel, H. Langmuir 1993, 9, 1361-1369. (13) Knoll, W. Mater. Res. Soc. Bull. 1991, 16, 29-39.
Boncheva et al. thus be calculated, given an appropriate layer refractive index and known relation between the layer density and refractive index. For the evaluation of our results we used a refractive index of 1.45;14 thus an angle shift of 0.1° corresponds to an average layer thickness of 6.4 Å. Sample Preparation for the SPR Measurements. Two different protocols were used for the LP/lipid layer formation: (i) self-assembly from mixed LP/lipid vesicles12 and (ii) horizontal Langmuir-Scha¨fer transfer.15 For the self-assembly experiments, glass slides (9 cm2, glass type SF10, n ) 1.730 ( 0.005, Guinchard, Yverdon-les-Bains, Switzerland) were cleaned by ultrasonication in detergent (Hellmanex, Hellma, Mu¨llheim, Germany) and water. Chromium and gold films (thickness 4 and 45 nm, respectively) were consecutively evaporated on a slide in a vacuum chamber at 5 × 10-6 mbar. The sample was immediately immersed in 1 mg/mL solution of tetradecanethiol in ethanol for at least 2 h. After rinsing in methanol and drying in air, the sample plate was optically matched to the base of the prism. It was pressed onto a Teflon support to form a reaction cell of 600 µL. A lipid or LP/lipid layer was formed on the alkanethiol monolayer by vesicle self-assembly. The formation of this layer was monitored in a time scan at a fixed angle of incidence. The subsequent angle scan gave an angle shift which allowed determination of the optical film parameters. For the Langmuir-Scha¨fer transfer experiments, metal layers were evaporated directly onto the base of the prism, which was incubated in a thiol solution as above. After being rinsed with methanol, the prism was lowered horizontally through a preformed LP or mixed LP/lipid monolayer at a surface pressure of 30 mN/m. This lateral pressure was chosen to be similar to that of the corresponding bilayer system.16 The Langmuir trough used in these experiments was designed to form the reaction cell together with the base of the prism. The transferred layer was thus protected from all possible contact with air. The binding of monoclonal antibodies to the immobilized antigen was followed by the change of the reflected light intensity at a fixed angle of incidence (53°). The cell content was exchanged several times with the corresponding Ab solution in order to avoid possible depletion of the solution. Formation of Small Unilamellar Vesicles. Two different protocols were used. In the alcohol injection method, used for the SPR experiments, appropriate volumes of the lipid and LP stock solutions were mixed in the desired molar ratio, dried in a stream of nitrogen, and redissolved in methanol to give a final LP concentration of 15 mM. This sample was then injected into buffer to give a final LP concentration of 150 µM and ultrasonified in a bath type sonicator at 4 °C for 3-5 min until a clear vesicle suspension was obtained. The resulting small unilamellar vesicles had a mean diameter of 30 nm and were able to specifically bind the monoclonal antibodies, as shown by cryoelectron microscopy. In the hydration method used for the CD experiments, typically 50-100 µg of LP and the corresponding amount of lipid for a LP/lipid molar ratio of 1:10 were mixed in an Eppendorf vial and dried down in a stream of nitrogen. The resulting dry membranes were dissolved to give a final concentration of 380 µM in 50 µL of Tris-EDTA buffer (1 mM Tris-HCl and 1 µM EDTA, pH 7.4) and ultrasonified as above.
Results and Discussion Secondary Structure of the LP. In order to obtain information about the LP structure in a lipid environment, we investigated the conformation of the peptide moiety in lipid membranes by CD and ATR-FTIR spectroscopy. Although for these measurements the LP has been incorporated in vesicles and dry multilayers, the results should give relevant information about its structure when organized in lipid monolayers at the water or gold surfaces. Figure 2 shows CD spectra of LP in POPC and DPPC vesicles at room temperature, i.e., in lipid membranes in (14) Pethig, R. Dielectric and Electronic Properties of Biological Materials; John Wiley: New York, 1979. (15) Langmuir, I.; Schaefer, V. J. J. Am. Chem. Soc. 1938, 60, 13511360. (16) Jones, M. N.; Chapman, D. Micelles, monolayers and biomembranes; Wiley-Liss, Inc.: New York, 1995.
Lipopeptide Layers
Langmuir, Vol. 12, No. 23, 1996 5639
Figure 2. CD spectra of the LP in methanol solution (filled symbols) and incorporated in phospholipid vesicles at a molar ratio LP/lipid ) 1/10 (open symbols). The peptide concentration was 380 µΜ, the temperature was 20 °C, and the path length of the cuvette was 0.1 mm. Table 1. Secondary Structure of Pure LP, LP/Lipid Preparations, and the Corresponding VP1 Peptide Segment secondary structure elements (%)b sample
R-helix β-strand β-turn undefined
Figure 3. ATR-FTIR spectra of dry LP/POPC multilayers at a molar ratio of 9/1. The best-fitted individual bands (thin solid lines), their sum (thick solid line), and the experimental points (open circles) are shown. The individual simulated bands were offset by -0.005 absorption unit in order to better distinguish them from their sum and the experimental data. The positions of the individual bands were deduced from the second-derivative spectra. The bands were assigned according to published references11,18,19 as follows: 1616 ( 2 cm-1, β-strand; 1631 ( 2 cm-1, β-strand; 1644 ( 2 cm-1, turn/β-strand; 1659 ( 2 cm-1, R-helix/disordered structures; 1684 ( 2 cm-1, turn/bend; 1728 ( 2 and 1740 ( 2 cm-1, lipid CdO vibrations.
the fluid or gel phase, respectively. The nearly identical spectra were evaluated in terms of percentages of different secondary structural elements as listed in Table 1. All different structural elements taken into consideration in the analysis were found to contribute significantly to the CD spectra, with a slight domination of the β-structures. For comparison with already published data17 we present also a CD spectrum of pure LP dissolved in methanol. In our case, the R-helix content was found to be higher in the organic solution than in the lipid vesicles. Similar results were obtained from the ATR-FTIR measurements. The features of the Amide I spectral region of LP in dried POPC multilayers (Figure 3) were observed also for LP in other phospholipid membranes. The relatively broad Amide I region was resolved into individual bands which were assigned to particular peptide secondary structures, as summarized in the legend of Figure 3. Two bands which arise from β-structures were resolved at 1616 ( 2 and 1631 ( 2 cm-1. There are other bands which confirm the presence of β-strands, as well as turns: 1644 ( 2 cm-1 (turn/β-strand), 1684 ( 2 cm-1 (turn/ bend). The feature at 1659(2 cm-1 indicates the presence of helical structures. Bands arising from R-helical struc-
tures are typically centered around 1657-1658 cm-1.20 A shift to higher wavenumbers, as in the present case, has been reported for distorted R-helices21 or 310-helices.22 The lipid chain CdO and CH stretching vibrations of pure LP multilayers were observed at frequencies characteristic for disordered lipid layers indicating that the palmitoyl chains in pure LP multilayers exhibit low conformational order. In summary, the secondary structure of the peptide part of the LP in lipid membranes as determined by CD and FTIR is in accord with the structure of this particular peptide segment in the native βG-βH loop in the capsid protein VP1 as determined by X-ray diffraction6 (Table 1). All conformational motifs present in the native viral protein were found as well in the synthetic peptide both in dry multilayers and in vesicles, of pure LP and mixed with lipids preparations. The difference in the β-sheet content in the whole virus crystals and in the LP preparations is understandable considering the different components forming the β-strands in the two cases. In the former one,6 the residues 144-146 of the VP1 form a strand of β-sheet, overlying residues 80-82 in strand C of VP2sinteraction which is absent in the case of the LP. Molecular Dimensions and Mixing at the Air/ Water Interface. Monolayer experiments combined with fluorescence microscopy were performed in order to obtain information about the molecular dimensions of the LP, its mixing behavior with different lipids, and its binding to monoclonal antibodies. The lipids used in these studies were chosen for the different phase behavior of their monolayers (POPC and DPPC) or the different charge of their headgroups (POPC and POPG). A typical surface pressure/area isotherm of the pure LP is shown in Figure 4. The collapse pressure of the layer was 48 mN/m. This pressure corresponds to an area of
(17) Krug, M.; Folkers, G.; Wiesmu¨ller, K.-H.; Freund, S.; Jung, G. Biopolymers 1989, 28, 499-512. (18) Byler, D. M.; Susi, H. Biopolymers 1986, 25, 469-487. (19) Frey, S.; Tamm, L. Biophys. J. 1991, 60, 922-930.
(20) Surewicz, W. K.; Mantsch, H. H.; Chapman, D. Biochemistry 1993, 32, 389-394. (21) Rothschild, K. J.; Clark, N. A. Biophys. J. 1979, 25, 473-488. (22) Kennedy, D. F.; Crisma, M.; Toniolo, C.; Chapman, D. Biochemistry 1991, 30, 6541-6548.
POPC vesiclesa DPPC vesiclesa LP solutiona peptide segment 135-154 of VP1 in crystalized virus particles
23 27 40 30
38 36 30 15
26 25 20 20
13 12 11 35
a Spectra and experimental conditions from Figure 2. b The standard deviation for the CD data is (5%; for analysis see Materials and Methods. The secondary structure in the crystallized virus particles are derived from ref 6.
5640 Langmuir, Vol. 12, No. 23, 1996
Boncheva et al.
Figure 4. Surface pressure-area isotherm of an LP monolayer spread on phosphate buffer, 5 mM, pH 7.4; temperature 20 ( 2 °C; compression speed 0.05-0.1 nm2 molecule-1 min-1.
0.77 nm2 per molecule. At pressures higher than 35 mN/m the film showed high viscosity and considerable hysteresis in the isotherm on expansion. The molecular area at a pressure of 30 mN/m (used for the LS transfers) was 1.5 ( 0.2 nm2. Isotherms of unlabeled and NBD-labeled LP were identical. During the compression, there was no indication of a first-order phase transition, as observed, for instance, with the phospholipid DPPC. LP layers doped with 2 mol % NBD-PE were observed under the fluorescence microscope during compression from 0 to 40 mN/m. This fluorescence probe is known to partition preferentially into the fluid phase of lipid monolayers. Homogeneous distribution of the probe fluorescence was observed over the whole surface pressure range. Mixed monolayers of LP and lipids were studied by fluorescence microscopy using the NBD-labeled LP. Upon compression of mixed NBD-LP/POPC layers (LP content 1-2 mol %), homogeneous NBD fluorescence was observed over the studied pressure range (0-35 mN/m). Upon compression of DPPC monolayer doped with 1.5 mol % NBD-LP up to a pressure of 10 mN/m (i.e., in the gaseous and the liquid-expanded state of the mixed layer), homogeneous distribution of the NBD fluorescence was observed. Above this pressure, we could follow the appearance and development of dark, solid-phase DPPC domains. In all stages of the compression the LP-label fluorescence was detected only in the liquid-analogous phase. The presence of the four positively charged amino acid residues in the peptide at pH 7.4 was expected to result in different molecular packing in uncharged compared to negatively charged lipid monolayers. For these studies we chose the phospholipids POPC and POPG with net charge per molecule of 0 and -1, respectively. At this pH and room temperature they form liquid-analogous monolayers. Isotherms of LP and lipids mixed in different molar ratios were measured on buffers of low (5 mM phosphate) and high (5 mM phosphate and 100 mM KCl) ionic strength as a subphase. The mean area per molecule of the LP/ POPC monolayers coincided (within the experimental error) with the linear interpolation between the molecular areas of the pure layers (results not shown). The mean area per molecule in the LP/POPG mixtures was constant and considerably lower than the linear interpolation between the molecular areas of the pure systems up to a stoichiometry of approximately 1:1. It increased drasti-
Figure 5. Fluorescence micrographs of a mixed NBD-LP/DPPC (2 mol %) monolayer at the water surface at a pressure of 14 mN/m after a 1 h incubation with Rhodamine-X-labeled antibodies at a concentration of 5 × 10-8 M, with the NBD (A) and the Rhodamine (B) filters. The bar corresponds to 50 µm.
cally for the higher molar ratios but remained unchanged upon increasing the ionic strength of the subphase. Since the molecular packing of the charged lipid layers was not affected by the ionic strength, electrostatic interactions between the molecules should not play any essential role. Since the two lipids differed also in the size of their headgroups, it is possible that the smaller POPG headgroup matches the hydrophilic LP part better than the relatively bulky PC, i.e., steric rather than electrostatic effects are important for the closer packing of mixed LP/ POPG compared to LP/POPC monolayers. Ab Binding to LP-Containing Langmuir Monolayers Observed by Fluorescence Microscopy. An important issue for characterization of Ab binding is the distinction between specific and nonspecific binding of the analyte. The mixed monolayers containing DPPC were found to be ideally suited for this purpose since phase separation leads to antigen-rich and antigen-free regions at the air/water interface. Thus Ab binding to these two regions could be studied simultaneously using differently labeled antibodies and lipopeptide. In addition, indirect ELISA tests showed that both the labeled and unlabeled compounds yielded binding constants in the range of 3 × 107 M-1. Rhodamine-X labeled antibodies were injected under such preformed monolayers to give a final subphase concentration of 5 × 10-8 M and the binding reaction was followed by the appearance of Rhodamine-X fluorescence at the interface. Figure 5 shows the same
Lipopeptide Layers
domain of the monolayer under the NBD (A) and the Rhodamine-X (B) filters of the microscope after 1 h of incubation with the antibodies. Homogeneous Rhodamine-X fluorescence was observed only in the liquidanalogous phase of the layer (where the LP was confined), thus demonstrating the high specificity of the binding reaction. No Rhodamine-X fluorescence was detected when pure DPPC monolayers were incubated with antibodies under the same conditions. Supported LP-Containing Lipid Membranes. The LP-containing monolayers were formed on top of thioalkane monolayers, self-assembled on gold. Several methods have been described for the formation of similar antigen-containing layers in this configuration: selfassembly from aqueous23 or organic solutions,24 detergent solutions,12 vesicles,2 or micelles.2 In the present study we compared the effectiveness of two of these methods: self-assembly of lipid vesicles varying in the LP/POPC composition, and horizontal, Langmuir-Scha¨fer transfer of a preformed POPC/LP monolayer onto a hydrophobized support. The first method resulted in considerable data scattering when the POPC/LP layer thickness was studied as a function of the antigen content in the initial solution for the vesicle preparation (data not shown). It is known that the composition of the mixed layer does not necessarily correspond to that of the initial solution or of the vesicles used and varies strongly with the history of the sample preparation: a similar problem has been encountered for layers self-assembled from lipid/detergent solutions,12 coadsorption of thiols on gold25 and immobilization of antigenic peptides on gold.26 In addition, the adhesion of intact vesicles to the supporting surface leading to increased mass coverage cannot be excluded.27 In contrast, the Langmuir-Scha¨fer method proved to be very effective for building supported monolayers with strictly predefined LP composition, but nonspecific antibody binding to such layers was significant for the higher (>10-7 M) Ab concentrations, possibly due to layer defects resulting from the transfer. Therefore, the dependence of the layer thickness and the Ab binding to the supported layers versus their composition were studied on layers transferred by the Langmuir-Scha¨fer technique, while binding isotherms were measured on self-assembled layers. Layer Thickness vs Composition. The thickness of the transferred LP/POPC layers (as shift of the resonance angle) is plotted as a function of Langmuir monolayer composition in Figure 6. The layer thickness increased linearly with LP content up to 70 mol % LP in the layer. For pure LP layers a maximum angle shift of 0.56 ( 0.05° was obtained, which corresponds to a layer thickness of 3.6 ( 0.3 nm using a refractive index of 1.45. These results were independent of the ionic strength of the buffer. In order to compare the actual layer thickness with that expected from the Langmuir monolayer’s composition, we used the following assumptions proposed elsewhere.26 The mixed LP/lipid monolayers were considered to be composed of two optically independent layers: one layer of hydrocarbon chains with a constant thickness of 1.9 ( 0.3 nm (∆θ ) 0.3°) and a second peptide-containing layer whose thickness varied with the composition. Using a typical (23) Spinke, J.; Liley, M.; Guder, H.-J.; Angermaier, L.; Knoll, W. Langmuir 1993, 9, 1821-1825. (24) Florin, E.-L.; Gaub, H. E. Biophys. J. 1993, 64, 375-383. (25) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7164-7175. (26) Duschl, C.; Se´vin-Landais, A.-F.; Vogel, H. Biophys. J. 1995, 70, 1985-1995. (27) Contino, P. B.; Hasselbacher, C. A.; Ross, J. B. A.; Nemerson, Y. Biophys. J. 1994, 67, 1113-1116.
Langmuir, Vol. 12, No. 23, 1996 5641
Figure 6. Angle shift upon LP/POPC monolayer transfer vs Langmuir monolayer composition. The solid line represents the calculated layer thickness for each composition (see the text). An angle shift of 0.1° corresponds to a thickness of 0.64 nm, using a refractive index of 1.45.
Figure 7. Angle shift due to monoclonal Ab binding to LP/ POPC transferred monolayers vs the Langmuir layer’s composition. The Ab concentration was 1 × 10-7 M. Nonspecific Ab binding resulted in an angle shift of 0.05 ( 0.02° for all layer compositions. An angle shift of 0.1° corresponds to a thickness of 0.64 nm, using a refractive index of 1.45.
value for the peptide density (1.37 g cm-3 28), the mass of the peptide molecule (4.4 × 10-21 g) and the experimentally determined molecular area (1.5 nm2), we calculated the expected thickness of the peptide-containing layer for each composition, assuming a refractive index of 1.45 (Figure 6, solid line). For the mixed monolayers the calculated values correspond reasonably well to the experimentally determined layer thicknesses. Ab Binding vs Layer Composition. The amount of Ab binding as a function of Langmuir monolayer composition was studied for LP-containing POPC layers (Figure 7). At a monoclonal antibody concentration of 1 × 10-7 M (well above the value of 1/Kas, see below) the thickness of the Ab layer increased with antigen content of the layer up to about 60 mol % LP, where it reached a saturation value of 4.8 ( 0.3 nm (0.75 ( 0.05°). In order to estimate the nonspecific Ab binding, layers with different antigen content were incubated with (28) Gennis, R. B. Biomembranes: Molecular Structure and Function; Springer-Verlag, Inc.: New York, 1989.
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Figure 8. Binding of the monoclonal antibody on a selfassembled LP/POPC layer of thickness 0.44 ( 0.3 nm. The best Langmuir-equation fit to the data (the solid line) yields an apparent association constant Kas of 1.9 × 107 M-1. An angle shift of 0.1° corresponds to a thickness of 0.64 nm, using a refractive index of 1.45.
polyclonal human and rabbit IgGs at the same concentrations as above; the monoclonal antibody binding to pure lipid layers was also tested. In all investigated cases the nonspecific protein binding was within the experimental error of 0.05°. The experimental data shown in Figure 7 can be recalculated in terms of an average surface density of the bound antibodies. We found it reasonable to use the same refractive index (n ) 1.45) and molecular density (1.37 g cm-3) for the monoclonal antibodies as for the peptide part of the LP. At the maximal value of the antibody layer thickness at about 60 mol % LP, a maximal surface density of one antibody molecule per 40 nm2 was calculated. An antibody of the immunoglobulin family has an elongated Y-shaped structure with typical minimal and maximal cross sectional areas of 45 and 90 nm2, respectively.29,30 According to our experimental data, the antibodies bind to lipid layers containing at least 60 mol % LP with the maximal surface density theoretically possible. Binding Isotherms. Binding experiments were performed on self-assembled and Langmuir-Scha¨fer layers of different LP content with Ab concentrations in the range 10-9-10-6 M. Here we discuss only the general features of the binding isotherms. Figure 8 represents a typical binding isotherm measured on a self-assembled layer of thickness 4.4 ( 0.3 nm. Using a Langmuir isotherm to fit the data, an apparent association constant Kas ) 1.9 × 107 M-1 was obtained. Figure 8, however, also shows that the experimental data are not perfectly described by this simple binding model. This behavior was observed for all LP-containing monolayers and all LP densities studied. Conclusions Our investigations have revealed three major points: (i) The peptidic portion of the LP under investigation adopts, both in pure lipopeptide and in mixed lipid layers, an average conformation comparable to that on the surface (29) Valentine, C. R.; Green, N. M. J. Mol. Biol. 1967, 27, 615-617. (30) Amzel, L. M.; Poljak, R. J. Annu. Rev. Biochem. 1979, 48, 961997. (31) Subramaniam, S.; Seul, M.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 1169-1173.
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of intact viral particles. One might speculate that such lipid monolayers serve for polar polypeptides as inert, structure-conserving surfaces and are therefore responsible for the excellent carrier and adjuvant properties of lipopeptides in antibody production. (ii) The LP forms mixed lipid monolayers with phospholipids in the fluid lipid state at any desired composition, either on the surface of water or of hydrophobic solid supports. (iii) The peptidic portion in these lipid monolayers is fully accessible for specific antibody binding. It is of particular importance in this context that such mixed lipid-lipopeptide surfaces perfectly suppress nonspecific protein adsorption. These exceptional properties of the supported lipid monolayers described are a consequence of their formation by self-organization based on the noncovalent interactions of lipids and lipopeptides, with each other as well as with the support. Thus the formation of defects with accessible hydrophobic surfaces is avoided by perfect intermolecular assembly due to the lateral mobility of the molecular lipid constituents. This, in turn, suppresses nonspecific protein adsorption. Furthermore, peptide or protein molecules attached to the lipid monolayer by lipid anchors are flexible enough to adopt a suitable conformation for specific molecular interactions with corresponding partners in the surrounding aqueous environment. Since they can be formed spontaneously by self-assembly on practically any transducer surface, supported lipid layers are ideally suited to direct in a controlled manner polar peptide moieties on surfaces. This may be useful for the investigation of molecular interactions at interfaces using novel surface sensitive techniques such as SPR, integrated optics, and scanning-probe microscopy and for the development of novel analytical tools. We believe that the supported lipid layers presented in this work are of particular importance for the reconstitution of biomolecular interactions of cell surfaces to biosensor applications. Nomenclature Ab, monoclonal antibodies against the peptide part of the lipopeptide ATR-FTIR, attenuated total reflection Fourier transform infrared CD, circular dichroism DPPC, 1,2-dipalmitoylphosphatidylcholine LP, lipopeptide NBD-LP, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole labeled lipopeptide Pam3Cys, N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cystein Pam3CysSerSer, N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteinylserylserin POPC, 1-palmitoyl-2-oleoylphosphatidylcholine POPG, 1-palmitoyl-2-oleoylphosphatidylglycerol SPR, surface plasmon resonance VP1 (135-154), amino acid sequence 135-154 from the capsid protein VP1 of the foot-and-mouth disease virus, subtype O1 Kaufbeuren
Acknowledgment. We thank Dr. Martha Liley for critically reading the manuscript. This work was supported by the Swiss National Science Foundation Priority Programme on Biotechnology, Grant 5002-35180, to H.V. LA9605753
