Langmuir 2004, 20, 7901-7903
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Controlled Immobilization of Membrane Proteins to Surfaces for Fourier Transform Infrared Investigations Per Rigler,† Wolf-Peter Ulrich, and Horst Vogel* Laboratoire de Chimie Physique des Polyme` res et Membranes, Ecole Polytechnique Fe´ de´ rale de Lausanne, CH-1015 Lausanne, Switzerland Received April 20, 2004. In Final Form: July 9, 2004 We show that it is possible to immobilize membrane proteins uniformly and reversibly as self-assembled (sub)monolayers on nitrilotriacetic acid-covered sensor surfaces via hexahistidine sequences present either in the protein or in lipid membranes. Fourier transform infrared spectra of such self-assembled (sub)monolayers deliver important structural information of the membrane proteins and are suited to screen the function of cellular receptors.
Results Membrane proteins, making up a third of the human proteome, fulfill central biological functions1 and as a consequence are important targets for therapeutic compounds.2 There is a strong demand for powerful methods to study the structure and function of mammalian membrane proteins which are difficult to express in large enough quantities for conventional structure-elucidating methods such as NMR spectroscopy, X-ray diffraction, and electron microscopy. Here, we report on a novel strategy to form selfassembled monolayers (SAMs) of membrane proteins and investigating them by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. Important results have earlier been obtained from ATR-FTIR on peptides in single monolayers,3,4 but this report shows the feasibility of using ATR-FTIR to immobilize and characterize monolayers of large membrane proteins. This spectroscopy offers unique possibilities to obtain information on the secondary structure, orientation, and water accessibility of membrane proteins if the proteins are properly immobilized on an ATR sensor surface. However, the potential of this technique has not been fully exploited yet because the proteins of interest are usually immobilized in the form of dried multilayered membranes and then are rehydrated for FTIR spectroscopic investigation. This procedure is affected with a number of severe limitations: (i) The control over the functional integrity of the immobilized proteins is lost during the drying process; (ii) the multilayered structure of the supported membranes may prohibit the full and fast accessibility of the proteins which is a prerequisite for investigating molecular interactions such as ligand binding to receptors or interaction of proteins playing a role in cellular signaling; (iii) the high protein consumption, usually in the 10-100-mg range,5,6 excludes the investigation of important mammalian receptor proteins which are gen* To whom correspondence should be addressed. E-mail:
[email protected]. Tel.: +41-21-6933155. Fax: +41-21-6936190. † Present address: Physikalische Chemie, Universita ¨ t Basel, Klingelbergstrasse 80, CH-4056 Basel, Switzerland. (1) Berg, J.; Tymoczko, J.; Stryer, L. Biochemistry, 5th ed.; WH Freeman: New York, 2002. (2) Drews, J. Science 2000, 287 (5460), 1960-1964. (3) (a) Frey, S.; Tamm, L. K. Biophys. J. 1991, 60 (4), 922-930. (b) Axelsen, P. H.; Kaufman, B. K.; McElhaney, R. N.; Lewis, R. N. Biophys. J. 1996, 69 (6), 2770-2781. (4) Flach, C. R.; Prendergast, F. G.; Mendelsohn, R. Biophys. J. 1996, 70, 539-546.
erally available only in much smaller amounts. Lipid membranes and membrane proteins have been immobilized on solid supports using different strategies mostly for biosensor applications.7-12 In previous work, we have shown the reversible immobilization of a polyhistidine peptide to ATR sensor surfaces covered with nitrilotriacetic acid (NTA) SAMs.13 Advantage was taken of the transition-metal-mediated specific interaction between NTA on the sensing surface and the polyhistidine sequence. In the present report, we extend this approach to the immobilization of two prototypic ligand-gated ion channels, the 5-hydroxytryptamine type 3 receptor (5-HT3R) and the nicotinic acetylcholine receptor (nAChR). Ligand-gated ion channels play a central role in cellular signaling and are important drug targets. Here, they serve as representative examples of important mammalian cell membrane proteins. The proteins were immobilized in two different ways: detergent-solubilized 5-HT3R via engineered hexahistidine sequences and lipid membrane reconstituted nAChR via hexahistdine-bearing lipids (Figure 1). In this way, uniform (sub)monolayers are achieved by binding the polyhistidine sequences to ATR supports previously modified with nitrilotriacetic groups. 5-HT3R and nAChR in D2O. Mutant 5-HT3Rs comprising N-terminal hexahistidine sequences have been shown to immobilize uniformly and reversibly on NTAcovered glass surfaces.14 Figure 2 shows the ATR-FTIR spectrum of 5-HT3R carrying a hexahistidine sequence at its N-terminus (solid line) and the ATR-FTIR spectrum (5) Go¨rne-Tschelnokow, U.; Strecker, A.; Kaduk, C.; Naumann, D.; Hucho, F. EMBO J. 1994, 13 (2), 338-341. (6) Gerwert, K.; Souvigner G.; Hess B. Proc. Natl. Acad. Sci. U.S.A. 1990, 87 (24), 9774-9778. (7) Radler, U.; Mack, J.; Persike, N.; Jung, G.; Tampe´, R. Biophys. J. 2000, 79 (6), 3144-3152. (8) (a) Zhao, J.; Tamm, L. K. Langmuir 2003, 19 (5), 1838-1846. (b) Groves, J. T.; Dustin, M. L. J. Immunol. Methods 2003, 278 (1-2), 1932. (9) Michel, R.; Reviakine, I.; Sutherland, D.; Fokas, C.; Csucs, G.; Danuser, G.; Spencer, N. D.; Textor, M. Langmuir 2002, 18 (22), 85808586. (10) (a) Heyse, S.; Stora, T.; Schmid, E.; Lakey, J. H.; Vogel, H. Biochim. Biophys. Acta 1998, 1376 (3), 319-338. (b) Bieri, C.; Ernst, O. P.; Heyse, S.; Hofmann, K.-P.; Vogel, H. Nat. Biotechnol. 1999, 17 (11), 1105-1108. (11) Naumann, R.; Jonczyk, A.; Kopp, R.; Vanesch, J.; Ringsdorf, H.; Knoll, W.; Graber, P. Angew. Chem., Int. Ed. Engl. 1995, 34 (18), 20562058. (12) Zhdanov, V. P.; Keller, C. A.; Glasmastar, K.; Kasemo, B. J. Chem. Phys. 2000, 112 (2), 900-909. (13) Rigler, P.; Ulrich, W. P.; Hoffmann, P.; Mayer, M.; Vogel, H. ChemPhysChem 2003, 4 (3), 268-275.
10.1021/la049002d CCC: $27.50 © 2004 American Chemical Society Published on Web 08/10/2004
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Figure 1. Schematic view of surfaces covered with a NTA SAM for immobilization of (A) a hexahistidine-comprising membrane protein in detergent and (B) a membrane protein in a lipid bilayer comprising hexahistidine-bearing lipids.
Figure 3. (A) ATR-FTIR spectra of nAChR in lipid membranes containing hexahistidine lipids immobilized on a NTA-SAM in H2O (solid line) and multilayers of nAChR from native membranes (dashed line) and (B) the corresponding difference ATR-FTIR spectrum (nAChR, submonolyer - nAChR, multilayer). Band assignments: lipid carbonyl stretch at 1735 cm-1, amide I at 1654 cm-1, amide II at 1549 cm-1.
Figure 2. Detergent-solubilized 5-HT3R comprising a Nterminal hexahistine sequence and nAChR reconstituted in membranes comprising hexahistidine lipids were immobilized to NTA-SAMs. The ATR-FTIR spectra of 5-HT3R (solid line) and nAChR (dashed line) in D2O show the amide I′ (1600-1700 cm-1) and II′ (1400-1500 cm-1) bands. Table 1. Secondary Structure of 5-HT3R and nAChR from Analysis of the Amide I and Amide I′ Bands secondary structure (%) 5-HT3Ra nAChRb nAChRc
R-helix
β-strand
β-turn
nonregular
36 34 37
39 37 37
11 14 12
14 15 14
a ATR-FTIR spectrum after complete hydrogen deuterium exchange. Protein solubilized in C12E9 detergent. b ATR-FTIR spectrum of nAChR reconstituted in supported lipid bilayers in H2O. c Transmission FTIR spectrum of nAChR in multilayers of native membranes.
of nAChR immobilized via hexahistidine-bearing lipids on NTA SAMs (dashed line) in D2O. The amide I′ and amide II′ bands at 1644 and 1455 cm-1 agree well with ATR-FTIR spectra of nAChR obtained elsewhere. The secondary structure of the 5-HT3R determined by curve fitting the amide I′ band of Figure 2 consists of 36% R-helix, 39% β-strand, 11% β-turn, and 14% nonregular structures; very similar results were obtained for the nAChR (Table 1) which is in complete agreement with already published data (see also Figure 4 in Supporting Information).15 Orientational effects do not have an impact on the determination of the secondary structure as can be seen from previously published polarized ATR-FTIR spectra of nAChRs in planar multilayer membranes; the parallel and the perpendicularly polarized ATR-FTIR spectra of (14) Schmid, E. L.; Tairi, A. P.; Hovius, R.; Vogel, H. Anal. Chem. 1998, 70 (7), 1331-1338. (15) Rigler, P.; Ulrich, W. P.; Hovius, R.; Ilegems, E.; Pick, H.; Vogel, H. Biochemistry 2003, 42 (47), 14017-14022.
the amide region differ in amplitude but are practically identical in their shape. In consequence, a band shape analysis of unpolarized amide I bands as done in the present communication delivers reliable secondary structures of the nAChR and the 5-HT3R.16 Comparison of FTIR Spectra of Different nAChR Preparations. Figure 3 shows ATR-FTIR spectra of nAChR (i) reconstituted in a single membrane, immobilized via hexahistine-bearing lipids on a NTA-SAM, in H2O buffer and (ii) in multilayers of native membranes in H2O. The amide I band of the single membrane is nearly identical to that of oriented multilayers, demonstrating that neither reconstitution into lipid membranes nor surface immobilization significantly changes the conformation of the nAChR (see also secondary structure evaluations in Table 1). Furthermore, these results are also in agreement with secondary structure data obtained from different biophysical techniques by different groups.15 Taken together, the method to immobilize monolayers of membrane proteins offers several important features for ATR-FTIR spectroscopy: (i) Detergent-solubilized and bilayer-inserted membrane proteins can be immobilized in a defined orientation on the sensor surface. (ii) The proteins are entirely solvated during the whole immobilization and subsequent measuring process. (iii) The monolayers of membrane proteins are fully and rapidly accessible, both to solvent exchange and to further molecular interactions. Thus, binding of ligands or other proteins can be studied easily. (iv) The chemical tethering of membrane proteins either directly or via lipid membranes allows the repetitive monitoring of molecular interactions of the receptor either for improving sensitivity or for studying sequential biochemical reactions. (v) The reversibility of the NTA-histidine tethering14,17 allows repetitive and fast immobilization of proteins on the sensor surface under strictly identical conditions for improving reliability and sensitivity of measurements. (16) Methot, N.; Ritchie, B. D.; Blanton, M. P.; Baenziger, J. E. J. Biol. Chem. 2001, 276 (26), 23726-23732.
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Although high signal-to-noise ratio spectra (over 1000) were readily achieved starting with small amounts (
