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Langmuir 2003, 19, 10304-10309
G-protein Interactions with Receptor-Derived Peptides Chemisorbed on Gold R. M. Petoral, Jr., A. Herland, K. Broo, and K. Uvdal* Laboratory of Applied Physics, Department of Physics and Measurement Technology, Linko¨ ping University, S-581 83 Linko¨ ping, Sweden Received June 13, 2003. In Final Form: August 21, 2003 Interactions between the functional bovine brain G-protein and receptor-derived peptides chemically adsorbed on gold surfaces are studied. The peptides are designed to mimic the third ic-loop (aa 361-373) of the Alpha 2a-adrenergic receptor (R2AR). These segments are linked to a surface using the thiol-gold chemistry, and the protein interaction studies are conducted to investigate the key function of recognition. The chemical composition and binding strength of the peptide monolayers onto a gold surface are characterized using X-ray photoelectron spectroscopy and infrared (IR) spectroscopy. Strong molecular binding of the adsorbates to the gold surface is attained, and the presence of amide-related IR vibrations verified the composition of the peptides. Bovine brain G-protein adsorption studies on these molecular monolayers are performed using the surface plasmon resonance technique. The arginine-rich peptide, which is a direct mimicry of the receptor, has a higher affinity for G-protein than the lysine-rich and alanine-rich derived peptides, showing that arginine residue has special importance for the G-protein interaction with the receptor.
1. Introduction Self-assembled monolayers (SAMs) of thiols and disulfides on gold are attractive systems for a variety of different applications. The interfacial phenomena of SAMs are increasingly studied as part of the new emerging field of biological surface science.1 One area of special interest is the ability to attach the receptor molecules to surfaces to study biomolecular interactions. The understanding of the specific interaction of these tailor-made surfaces with proteins is important in the field of biosensor technology.2 Studies have been conducted on some adsorbed polypeptides3-6 and peptides, using the gold thiol chemistry, to investigate protein interaction specificity.7 One of the largest families of membrane-bound receptors is the 7-helix transmembrane G-protein-coupled receptors (GPCRs). GPCRs are involved in cell signaling pathways that are essential for most physiological functions. The understanding of how the heterotrimeric guanine nucleotide-binding protein (G-protein) interacts with the receptor is interesting, to design intracellularly functioning drugs. The R and β subunits of the G-protein are believed to interact with the receptor,8 and these subunits have been considered as potential drug targets.9 Because of their functional importance, considerable efforts have been made to solve the structure of GPCRs, as well as to understand the mechanism of activation and the location of binding sites for the G-proteins.10,11 * Author to whom correspondence should be addressed. E-mail:
[email protected]. (1) Kasemo, B. Surf. Sci. 2002, 500, 656. (2) Wink, Th.; van Zuilen, S. J.; Bult, A.; van Bennekom, W. P. Analyst 1997, 122, 43R. (3) Deming, T. J. Adv. Mater. 1997, 9, 299. (4) Williams, A. J.; Gupta, V. K. J. Phys. Chem. B 2001, 105, 5223. (5) Higuchi, M.; Koga, T.; Taguchi, K.; Kinoshita, T. Langmuir 2002, 18, 813. (6) Williams, A. J.; Gupta, V. K. Thin Solid Films 2003, 423, 228. (7) Duschl, C.; Sevin-Landais, A.-F.; Vogel, H. Biophys. J. 1996, 70, 1985. (8) Taylor, J. M.; Jacob-Mosier, G. G.; Lawton, R. G.; Remmers, A. E.; Neubig, R. R. J. Biol. Chem. 1994, 296, 27618. (9) Holler, C.; Freissmuth, M.; Nanoff, C. Cell Mol. Life Sci. 1999, 55, 257.
The importance of the second and third intracellular loops, together with the C-terminal tail of the G-proteincoupled R2-adrenergic receptor (R2AR) as recognition sites for G-protein interactions and binding, have been studied.8,12-14 Several peptides have been derived from GPCRs, and peptide interaction studies with G-proteins have been made.8,15-19 Arginine residues present at the C-terminal region of the third intracellular (ic3) loop are suggested to have a specific role in G-protein recognition and activation.13 Recently, we have performed some significant work to probe the molecular interaction and recognition of bovine brain G-proteins and the arginines using the surface plasmon resonance (SPR) technique.20,21 Arginine is linked to either L-cysteine or cysteamine residues to enable oriented adsorption to the gold sensor/ surface. Both arginine-containing peptides have indicated the binding of intact G-protein. A higher affinity of G-protein to the arginine-cysteamine adsorbate than the arginine-L-cysteine adsorbates has been observed.21 In arginine-cysteamine adsorbates, the arginine is more available for protein adsorption, because of its orientation when adsorbed on the gold surface. Our previous work also noted the importance of the choice of buffer to keep the G-protein in a native conformation. (10) Scho¨neberg, T.; Schultz, G.; Gudermann, T. Mol. Cell. Endocrinol. 1999, 151, 181. (11) Eason, M.; Liggett, S. B. J. Biol. Chem. 1995, 271, 12826. (12) Dalman, H. M.; Neubig, R. R. J. Biol. Chem. 1991, 266, 11025. (13) Wade, S. M.; Scribner, M. K.; Dalman, H. M.; Taylor, J. M.; Neubig, R. R. Mol. Pharmacol. 1996, 50, 351. (14) Chung, D. A.; Wade, S. M.; Fowler, C. B.; Woods, D. D.; Abada, P. B.; Mosberg, H. I.; Neubig, R. R. Biochem. Biophys. Res. Commun. 2002, 293, 1233. (15) Higashijima, T.; Burnier, J.; Ross, E. M. J. Biol. Chem. 1990, 265, 14176. (16) Voss, T.; Wallner, E.; Czernilofsky, A. P.; Freissmuth, M. J. Biol. Chem. 1993, 268, 4637. (17) Varrault, A.; Nguyen, D. L.; McClue, S.; Harris, B.; Jouin, P.; Bockaert, J. J. Biol. Chem. 1994, 269, 16720. (18) Franzoni, L.; Nicastro, G.; Pertinhez, T. A.; Oliveiera, E.; Nakaie, C. R.; Paiva, A. C. M. J. Biol. Chem. 1999, 274, 227. (19) Hessling, J.; Lohse, M. J.; Klotz, K. N. Biochem. Pharmacol. 2003, 65, 961. (20) Uvdal, K.; Vikinge, T. Langmuir 2001, 17, 2008. (21) Petoral, R. M.; Uvdal, K. Colloids Surf., B 2002, 25, 335.
10.1021/la035046v CCC: $25.00 © 2003 American Chemical Society Published on Web 10/10/2003
G-protein Interactions with Peptides on Gold
In this study, we examined the functional bovine brain G-protein interaction with chemically adsorbed peptides, mimicking the ic3-loop of the receptor R2AR. A segment of the ic3-loop (aa 361-373) of the R2AR is used as a template in the design of G-protein binding peptides, with three glycines and a cysteine residue added to the C-terminal of the peptide sequence. These segments are linked to the gold surface through thiol coupling chemistry. Related peptides are also synthesized to investigate the importance of electrostatic forces in the receptor-Gprotein interaction processes. The composition and binding of the peptide monolayers on the surface is studied by X-ray photoelectron spectroscopy (XPS), complemented by IR spectroscopy. G-protein interaction with the synthesized peptides is studied using the SPR technique. 2. Experimental Section 2.1. Syntheses and Purification of Peptides. The peptides were synthesized on a Pioneer Peptide synthesizer (Perseptive Biosystems), using standard Fmoc chemistry. Cleavage of the peptide from the resin was performed using a trifluoroacetic (TFA) solution. The peptides were purified using reverse-phase high-performance liquid chromatography (HPLC) (Varian, Pro Star). The mobile phase consisted of 0.1% TFA in water (Milli-Q) and acetonitrile. After purification, the peptides were lyophilized and stored at -70 °C. The correct mass of the peptides was confirmed using matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (Voyager DE). 2.2. Sample Preparation. The gold substrates used were prepared via the electron beam evaporation of a 2000 Å thick layer of gold, at a rate of 10 Å/s onto clean single-crystal Si (100) wafers. Before evaporation of the gold film, the silicon wafers first were coated with a titanium layer 20-25 Å thick at a rate of 2 Å/s. The base pressure was always
