Langmuir 1993,9,1361-1369
1361
Protein Binding to Supported Lipid Membranes: Investigation of the Cholera Toxin-Ganglioside Interaction by Simultaneous Impedance Spectroscopy and Surface Plasmon Resonance Samuel Terrettaz,+ Thierry Stora,? Claus Duschl, and Horst Vogel' Imtitut de chimie physique 11, Ecole Polytechnique Fkdkrale de Lausanne, CH-1015 Lausanne, Switzerland Received November 10,1992.In Final Form: January 28,1993 The specificrecognition reaction between choleratoxin (ChTo)and the gangliosideG Mwas ~ investigated in situ on planar supported lipid layers using simultaneous surface plasmon resonance (SPR) and electrochemicalimpedance measurements. The lipid monolayers containing different amounts of G M ~ were formed on alkanethiol-coatedgoldsurfacesby a new lipiadetergent dilution technique. The formation of the layers was investigated using both SPR and impedancemeasurements. While the opticalproperties of the different lipid layers remained unchanged, the layer capacitance was seen to depend on the G M ~ to POPC molar ratio. The selective binding of ChTo to these layers was detected with high sensitivity by SPR. The comparatively small impedance changes associated with the protein adsorption suggest a high water content of the ChTo layers. The B5 subunit has been shown to interact similarly with these supported lipid layers. Introduction The characterization of surfaces and interfaces plays an important role in disciplines ranging from physics and chemistry to biology and ele~tronics.~-3 The technicallyoriented issues concern the engineeringof supramolecular structures with designed properties; these structures may have technological potential in optical and molecular electronics, for the development of novel sensor devices or for the modulation of interfacial properties (such as wettability, adhesion,tribology, and corrosion,to mention only a few). The techniques presently available for the construction of such systems include Langmuir-BlodgettKuhn (LBKI4and self-assembly (SA) methods: by which (starting from the properties of individualbuildingblocks) ordered, monomolecular layers can be formed on solid surfaces. By incorporating specificfunctional groups into such systems, it might be possible to design, in a 'bottomup construction", new hyperstructures with increasing complexity and multiple selectivities.2 In addition to the technological interest, the biological sciences are also frequently concerned with interfaces, primarily in relation to biologicalmembranes. These thin, sheetlike structures (ca. 100 8, in thickness), composed of lipid and protein molecules, fulfill a variety of functions of fundamental importance. The understanding of the structural and dynamic interactions between the various membrane components,as amolecular basis for the highly organized and complexfunctions of molecular membranes, is one of the great challenges in modern biology and biophysics. The topic of the present paper is to investigate the formation of lipid monolayers on hydrophobic solid
substrates. The supported lipid layers should be comprised of biological, surface receptor molecules, suitable for the selective binding of ligand molecules from the surrounding aqueous phase. These layers represent a simple model for biologicalmembranes. The ultimate goal is to develop lipid-based receptors on solid substrates, which might be useful both for the practical applications mentioned above as well as for studying the formation of organized molecular layers, the structure and dynamicsof the assembled,biological molecules,and the concomitant, basic mechanism of the ligand-receptor interaction at the lipid-water interface. The receptors of interest in the present context are, in general, amphipathic molecules, either lipid molecules themselves, or lipid-anchored peptides or proteins. Here, we have investigated the interaction of cholera toxin (ChTo) with monosialoganglioside (GM~) incorporated in planar, supported lipid monolayers on gold substrates. The GMl-ChTo couple is an attractive model systems for our present purposes, Le., the investigation of ligand-receptor interactions at solid-supported lipid membranes, for the following reasons: (i) the membrane receptor G Mis~a lipid moleculeand therefore can be easily incoporated into supported lipid monolayers; (ii) both the receptor and the ligand ChTo can be isolated from natural sources in relatively large quantities and are available in pure form; (iii)the receptor-ligand complex is chemically well characterized, which facilitates the interpretation of the experimental results on solid supports. ChTo, an enterotoxin of Vibrio cholerae, is composed of two subunits, A (M, 27 kDa) and B (M, 11.6 m a ) , with the stoichiometry ABS. The protein binds via the B components to specific receptors on the cell surface, the Both of these authors contributed equally to this work. G M gangliosides.6 ~ In the subsequent cascade reaction, * To whom correspondence should be addressed. the fragment A1 of the A subunit is translocated through (1) Swalen,J.D.;Allara,D.L.;Andrade,J.D.;Chandroas,E.A.;Garoff, the membrane of the host cell, where it activates the S.; Israelachvilli, J.; McCarthy, T. J.; Murray, R.; Pease, R. F.; Rabolt, adenylate cyclase. J. F.; Wynne, K. J.; Yu, H. Langmuir 1987, 3, 932. (2) Kuhn, H. Thin Solid Films 1989, 178, 1. Recently, the three-dimensional structure of the heat(3) Carter, F. L. Molecular Device Electronics; Marcel Dekker: New labile enterotoxin from Escherichia coli, a protein closely York, 1987. (4) Kuhn, H.; Mobius, D.; Bucher, H. In Physical Methods of Chemistry; Weissberger, A,,Rossiter, B. W., Eds.;Wiley: New York, 1972; Vol. I, Part 111. (5) Bain, C. D.; Whitesides, G . M. Angew. Chem. 1989,101/4,522.
(6) Fishman, P. H. In ADP-Ribosylating Toxins and G-Proteins: Insight into Signal Transduction; Moss,J., Vaughan, M., Eds.;American Society of Microbiology: Washington, DC, 1990.
0743-7463/93/2409-1361$04.00/00 1993 American Chemical Society
Terrettaz et al.
1362 Langmuir, Vol. 9, No. 5, 1993 related to ChTo, has been determined by X-ray diffraction. The structure of the A subunit and that of the B pentamer, as well as their relative arrangements, have been revealed at atomicresolution.' Furthermore, the structure of ChTo bound to lipid monolayers has been investigated by electron microscopy of two-dimensional crystals to a resolution of 15 A.a11 There remains the open question of how ChTo binds to the membrane and subsequently translocates the A1 subunit through the membrane. In the present work, we have developed a new method for forming a supported lipid layer which is distinguished by its simplicity. A thin gold film, evaporated on a glass plate, serves as a solid support. First, a layer of closelypacked hydrocarbon chains is coupled to the gold surface by SA of alkanethiols, which form covalent sulfur-gold bonds. The alkanethiol-covered gold surface is ideally suited for SA of a second molecular layer of amphipathic molecules (lipids in our present case). Two procedures will be investigated (i) starting from an aqueous solution of lipids in detergent, dilution to below the critical micellar concentration (cmc)of the detergent leads to the formation of a second, stable lipid layer on the hydrophobic alkanethiol surface;12(ii) lipid vesicles in contact with the supported alkanethiollayer form,under certain conditions, a second lipid layer. As both procedurescan be performed in aqueous solution under relative mild conditions, they are of general applicability for the reconstitution of biologically active receptor molecules, such as lipidanchored proteins. A somewhat related procedure has been described for the formation of lipid monolayers on silanized glass surfaces by a detergent dialysis method.13 The particular configuration of the system described allows the simultaneous application of surface plasmon resonance (SPR) spectroscopy and electrical impedance measurements in order to observe the formation of molecular assemblies at solid surfaces, as well as to investigatethe ligand-receptor interactions at these solidsupported layers. SPR is a surface-sensitive technique which detects changes of the optical parameters in the vicinty of a solid-liquid interface with high sen~itivity.'~ The technique has been used to study molecular assemblies on silver and gold surfaces, for example, the formation of supported lipid mono- and bilayers,15J6the interactions between antigens and antibodies," and the interactions between streptavidin and biotin.18 Electrical impedance measurements, particularly when performed at different frequencies, typically between and loll Hz,yield information on the dielectric properties of materials.19 Measurementsof the capacitanceand conductanceof lipid membranes have had great impact in revealing the basic, structural features of lipid bilayer structures.20 Recently this technique has been applied to the study of the lipid layers on solid supports, for example, the formation of (7)Sixma, T. K.; Pronk, S.E.;Kalk, K. H.; Wartna, E.S.; van Zanten, B. A. M.; Withold, L. B.; Hol, W. G. J. Nature 1991,351, 371. (8)Ribi,H. O.;Ludwig,D. S.;Mercer,K.L.;Schoolnik,G. K.; Kornberg, R. D. Science 1988.239.1273. (9) Ludwig, D. S.';Ribi, H. 0.; Schoolnik, G. K.; Kornberg, R. D. Proc. Natl. Acad. Sci. U.S.A. 1986,83,8585. (10)Reed, R. A.; Mattai, J.; Shipley, G. G. Biochemistry 1987,26,824. (11)Mosser, G.; Mallouh, V.; Brisson, A. J. Mol. Biol. 1992,226,23. (12) Lang, H.; Duschl, C.; GrPtzel, M.; Vogel, H. Thin Solid Films 1992,2101211,818. (13)Huang, L.Biochemistry 1985,24, 29. (14)Knoll, W. MRS Bull. 1991,16,29. (15)Rothenhiiusler, B.;Knoll, W. Nature 1988,337,6165. (16)Rothenhausler, B.;Duschl, C.; Knoll, W. Thin Solid Films 1988, 159,323. (17)Filgerstam, L. G.; Frostell-Karlsson, A,; Persson, B.; RGnneberg, I. J. Chromatogr. 1992,597,397. (18)Haussling, L.; Ringsdorf, H.; Schmidt, F.-J.; Knoll, W. Langmuir, 1991,7, 1837. (19)Macdonald, J. R.; Johnson, W. B. In Impedance Spectroscopy; Macdonald, J. R., Ed.; Wiley: New York, 1987;p 13. ~
lipid mono- and bilayers,12the selective binding of ions to layer-incorporated ionophores,2' and the absorption of peptides to such membrane surfaces.22
Materials and Methods Materials. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, purity >99% ), octanethiol, and hexadecanethiol (purity >95%) were purchased from Fluka; bovine brain monosialoganglioside (Ghll),bovine serum albumine (BSA), cholera toxin (ChTo) and cholera toxin B5 subunit were from Sigma; n-octyl j3-D-glucoside (octyl glucoside) was from Bachem and rac-l,2dioleoylglycero-3-phospho-l'-glycerol (DOPG) from Avanti. All other chemicals were obtained from Fluka and were of the highest quality available. The water used was purified via an ion exchanger purification train (Milli-Q system, Millipore). The buffer comprised 0.05 M Tris-HC1 (pH 7.5), 0.2 M NaC1, and 1 mM NazEDTA. The purity of the protein preparations was checked by sodium dodecyl sulfate (SDS) gel electrophoresis under reducing conditions in the case of the ChTo and by a nondenaturing gel in the case of the B5 subunits of ChTo. Three bands were observed at apparent molecular masses of ca. 7, 13, and 28 kDa for the ChTo, which correspond to the Al, B, and A2 subunits, respectively. The apparent molecular masses of the A1 and A2 subunits as detected by SDS electrophoresis differ slightly from those calculated from their molecular sequences, in accordance with similar observations by others.23 A major band, with an apparent relative molecular mass of ca. 50 kDa, was observed for the B5 subunit. According to published data,23,24the results indicate intact and apparently pure protein preparations of ChTo and ita pentameric form B subunit. The proteins were stored in buffer at 4 OC and used within 2 weeks. No change in the ~ electrophoretic behavior or in its binding activity to G Mwas observed during this period of time. Electrode Preparation. SFlO glass slides (Guinchard S.A., Switzerland) with an index of refraction n = 1.723were cleaned with chromicacid, and, in thecaseof the LBKtechnique described in the following section, were subsequently silanized with dimethyldichlorosilane (Fluka). A 12 X 5 mm2rectangular, 4-nmthick chromium layer was first vacuum-deposited (
