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Langmuir 2006, 22, 7260-7264
r2A-Adrenergic Receptor Derived Peptide Adsorbates: A G-Protein Interaction Study Cecilia Vahlberg, Rodrigo M. Petoral Jr., Carina Lindell, Klas Broo, and Kajsa Uvdal* DiVision of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linko¨ping UniVersity, SE-581 83 Linko¨ping, Sweden ReceiVed October 18, 2005. In Final Form: May 18, 2006 The affinity of R2A-adrenergic receptor (R2A-AR) derived peptide adsorbates for the functional bovine brain G-protein is studied in the search for the minimum sequence recognition. Three short peptides (GPR-i2c, GPR-i3n, and GPR-i3c) are designed to mimic the second and third intracellular loops of the receptor. X-ray photoelectron spectroscopy is used to study the chemical composition of the peptides and the binding strength to the surfaces. Chemisorption of the peptides to the gold substrates is observed. Infrared spectroscopy is used to study the characteristic absorption bands of the peptides. The presence of peptides on the surfaces is verified by prominent amide I and amide II bands. The interaction between the peptides and the G-protein is studied with surface plasmon resonance. It is shown that GPR-i3n has the highest affinity for the G-protein. Equilibrium analysis of the binding shows that the G-protein keeps its native conformation when interacting with GPR-i3c, but during the interaction with GPR-i2c and GPR-i3n the conformation of G-protein is changed, leading to the formation of aggregates and/or multilayers.
1. Introduction Development of biologically inspired materials with new properties and functionalities imitating naturally existing biological functions, by designing tailor-made surfaces as engineered nanostructures, is important in biosensor design and biomimetics.1 Surface immobilized bioactive peptides are useful in proteomics, drug discovery, and sensor applications.2-4 Membrane signal transduction, i.e., the process in which extracellular signals are converted into cellular responses, is often mediated by membrane-bound G-protein coupled receptors (GPCRs). Schematic structures of a GPCR spanning the membrane are shown in Figure 1. GPCR is a very large family of cell surface receptors that mediates cell signaling in many of our physiological processes and is of major interest as potential drug targets. The majority of the drugs used clinically in humans target these 7-helix transmembrane receptors.5 The possibility to use guanine nucleotide binding proteins (G-proteins) as drug targets has also been suggested.6 Binding of a ligand to the receptor is believe to induce conformational changes that enable the G-protein to interact with earlier nonexposed regions in the intracellular parts of the receptor.7 It is well-known that the intracellular loops are important for G-protein coupling and activation. As the heterotrimeric G-protein binds the receptor, it is activated by a GDP/GTP exchange in the GR subunit. The R subunit undergoes conformational changes that cause the GTP-GR complex and Gβγ to dissociate from each other. The two parts can separately interact with different effector proteins.6,7 Receptor-derived peptides have frequently been used to study receptor-G-protein interaction.8-11 A peptide mimicking the * Corresponding author. Fax: +46-13-288-969. E-mail:
[email protected]. (1) Kasemo, B. Surf. Sci. 2002, 500, 656. (2) Kamel, R.; Eftekhari, P.; Garcia, S.; Berthouze, M.; Berque-Bestel, I.; Peter, J.-C.; Lezoualc’h, F.; Hoebeke, J. Biochem. Pharmacol. 2005, 70, 1009. (3) Morohashi, K.; Yoshino, A.; Yoshimori, A.; Saito, S.; Tanuma, S.; Sakaguchi, K.; Sugawara, F. Biochem. Pharmacol. 2005, 70, 37. (4) Chow, E.; Wong, E. L. S.; Bo¨cking, T.; Nguyen, Q. T.; Hibbert, D. B.; Gooding, J. J. Sens. Actuators B 2005, 111-112, 540. (5) Flower, D. R. Biochim. Biophys. Acta 1999, 1422, 207. (6) Holler, C.; Freissmuth, M.; Nanoff, C. Cell. Mol. Life. Sci. 1999, 55, 257. (7) Hamm, H. E. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4819.
Figure 1. Schematic structures of a G-protein coupled receptor (GPCR) spanning the membrane.
carboxyl terminal part of the third intracellular loop (i3c) of an R2-adrenergic receptor (R2-AR) was shown to bind both the R and the β subunits.8 This peptide could also stimulate GTPase activity of the G-protein.8 Basic residues within the i3c region have been shown to be important for G-protein activation.9,10 Mutagenesis studies have shown that the second intracellular (8) Taylor, J. M.; Jacob-Mosier, G. G.; Lawton, R. G.; Remmers, A. E.; Neubig, R. R. J. Biol. Chem. 1994, 269, 27618. (9) Wade, S. M.; Lim, W. K.; Lan, K.-L.; Chung, D. A.; Nanamori, M.; Neubig, R. R. Mol. Pharmacol. 1999, 56, 1005. (10) Wade, S. M.; Scribner, M. K.; Dalman, H. M.; Taylor, J. M.; Neubig, R. R. Mol. Pharmacol. 1996, 50, 351. (11) Eason, M. G.; Liggett, S. B. J. Biol. Chem. 1996, 271, 12826.
10.1021/la052801r CCC: $33.50 © 2006 American Chemical Society Published on Web 07/14/2006
R2A-Adrenergic Receptor DeriVed Peptide Adsorbates
loop and the amino terminal part of the third intracellular loop of R2A-AR are important for G-protein coupling.11 We are studying surface immobilized molecules focusing on molecular recognition. Our aim is to find peptide sequences that selectively and with a high affinity interact with the G-protein. We have earlier investigated the binding of G-protein to a variety of molecules, such as amino acids, amino acid derivatives, and peptide sequences, to be used as sensor elements for the G-protein, e.g. L-cysteine, cysteamine, the dipeptide arginine-L-cysteine (Arg-Cys), and the related molecule arginine-cysteamine.12,13 Comparison between immobilized arginines as Arg-Cys and Argcysteamine showed the importance of molecular orientation.13 Recently, the interaction between functional bovine brain G-protein and synthetic receptor derived peptides was studied.14 The peptides were designed to mimic the third intracellular loop of the R2A-AR. Our aim was to investigate the significance of the presence of arginine in the receptor-derived peptide adsorbates. An exchange of the basic arginine residues to either basic lysine residues or nonpolar alanine residues significantly reduced the peptide adsorbates selectivity for G-protein.14 Our results showed that arginines in the ic3-loop have a specific role in the interaction with the G-protein. In this study we focus on the three different intracellular regions of the R2A-AR that earlier have been suggested to be of importance for G-protein coupling and/or activation. Three peptides were designed to mimic active sequences of the second (i2c) and third (i3n, i3c) intracellular loops. The peptides were chemisorbed onto gold surfaces using thiol chemistry. X-ray photoelectron spectroscopy (XPS) was used to study the binding strength between the peptides and the gold surfaces and the chemical composition of the peptides. Amide absorption bands characteristic for peptides were studied with infrared (IR) spectroscopy. Ellipsometry was used as a complementary technique. The interaction between the synthetic receptor-derived peptides (GPRi2c, GPR-i3c, GPR-i3n) and the G-protein was studied in realtime using surface plasmon resonance (SPR). 2. Experimental Section 2.1. Peptide Synthesis and Purification. The peptides were synthesized by Fmoc solid-phase peptide synthesis on a Pioneer peptide synthesizer (Perseptive Biosystems). Cleavage of the peptides from the solid support was performed in trifluoroacetic (TFA) solutions. Reversed-phase high-performance liquid chromatography (HPLC) (Varian, Pro Star) was used to purify the peptides. The mobile phase consisted of acetonitrile, water (MilliQ), and 0.1% TFA. The mass and purity of the peptides were confirmed by a matrix-assisted laser desorption/ionization time-of flight mass spectrometer (VoyagerDE STR, PerSeptive Biosystem). After purification, the peptides were lyophilized and stored at -76 °C until use. 2.2. Sample Preparation. Gold surfaces used for ellipsometry, XPS, and IRAS were prepared by using an electron beam evaporation system. Clean, single-crystal silicon (100) wafers were first coated with a 25 Å thick titanium film at a rate of 2 Å/s. The precoated wafers were then covered by a 2000 Å thick gold layer at a rate of 10 Å/s. The base pressure was below 5 × 10-9 Torr and the evaporation pressure below 2 × 10-8 Torr. Gold surfaces from Biacore AB were used for the surface plasmon resonance measurements. The gold surfaces were washed in a basic peroxide solution [MilliQ water:NH3 (25%):H2O2 (28%) (5:1:1)] at 80 °C for 5 min to remove (12) Uvdal, K.; Vikinge, T. P. Langmuir 2001, 17, 2008. (13) Petoral, R. M.; Uvdal, K. Colloids Surf. B: Biointerfaces 2002, 25, 335. (14) Petoral, R. M.; Herland, A.; Broo, K.; Uvdal, K. Langmuir 2003, 19, 10304.
Langmuir, Vol. 22, No. 17, 2006 7261 organic contaminations from the substrates before incubation and then carefully rinsed with MilliQ water. The incubation solutions were prepared with degassed MilliQ water and kept in darkness to reduce the risk of oxidation. The concentration of the solutions was 70 µM and the incubation time was longer than 36 h. After incubation, the surfaces were ultrasonicated in MilliQ water for at least 5 min and rinsed with MilliQ water, dried in nitrogen gas, and immediately placed in the instruments. Multilayer samples were prepared by drying concentrated droplets of peptide solution onto gold surfaces for the XPS measurements and a CaF2 window for transmission infrared spectroscopy measurements. 2.3. X-ray Photoelectron Spectroscopy. The XPS spectra were recorded on a VG spectrometer with unmonochromatized Mg KR photons (1253.6 eV) and a CLAM2 analyzer. The pressure in the analysis chamber was ∼10-10 mbar and the temperature ∼298 K. The measurements were based upon photoelectrons with a takeoff angle of 30° relative to the surface normal of the sample. The power of the X-ray gun was kept constant at 300 W. The XPSPEAK95 program (version 2.0) was used to calculate the chemical composition of the peptides from the peak areas and to analyze the peak positions. The binding energy scale of the monolayer spectra was aligned through the Au (4f7/2) peak at 84.0 eV. 2.4. Infrared Spectroscopy. The infrared reflection absorption spectra were recorded on a Fourier transform spectrometer (Bruker, model IFS66) with a mercury cadmium telluride (MCT) detector. The spectrometer has a grazing angle of incidence reflection accessory aligned at 85°. Liquid nitrogen was used to cool the detector before the measurements. The measurement chamber was continuously purged with nitrogen gas before and during the measurements, to reduce the contribution of water and carbon dioxide. The spectra were recorded by averaging 2000 interferograms at 2 cm-1 resolution. A three-term Blackmann-Harris apodization function was applied to the interferograms before Fourier transformation. The transmission infrared spectra were recorded with a Bruker IFS 66v spectrometer with a deuterated triglycine sulfate (DTGS) detector at 5 mbar pressure. The spectra were recorded by averaging 500 interferograms at 2 cm-1 resolution. 2.5. Ellipsometry. The thickness of the peptides was measured, with single wavelength (λ ) 632.8 nm) null ellipsometry. The measurements were performed on an automatic Rudolph Research AutoEl ellipsometer with a He-Ne laser light source, at an angle of incidence of 70°. The peptide film was assumed to be isotropic and transparent with a refractive index of n ) 1.5.15 The refractive index of clean gold surfaces was measured prior to incubation. The collected average values were after incubation used together with the refractive index of the ambient atmosphere, the refractive index of the peptide film, and the determined ellipsometric angles of the film to determine the thickness. The thickness of the peptide films was measured at five different positions on 7-11 samples for each peptide. 2.6. Surface Plasmon Resonance. The SPR measurements were performed on a Biacore 2000 instrument (Biacore AB). The functional bovine brain G-protein (Calbiochem) used in the SPR measurements contains a mixture of the following heterotrimeric G-proteins: G0R (∼4-5 µM), GiR-1 (∼1-2 µM), GiR-2 (∼1-2 µM), and GiR-3 (
