Mixed Monolayers to Promote G-Protein Adsorption - American

8474

Langmuir 2007, 23, 8474-8479

Mixed Monolayers to Promote G-Protein Adsorption: r2A-Adrenergic Receptor-Derived Peptides Coadsorbed with Formyl-Terminated Oligopeptides Luminita Savitchi Balau, Cecilia Vahlberg, Rodrigo M. Petoral, Jr., and Kajsa Uvdal* DiVision of Sensor Science and Molecular Physics, Department of Physics, Chemistry and Biology, IFM, Linko¨ping UniVersity, SE-581 83 Linko¨ping, Sweden ReceiVed NoVember 27, 2006. In Final Form: May 14, 2007 Pure and mixed monolayers of a synthetic peptide, GPR-i3n, derived from the third intracellular loop of the R2 adrenergic receptor and a shorter inactive oligopeptide, N-formyl-(Gly)3-(Cys) (called 3GC), were prepared on gold surfaces. The mixing ratio of the GPR-i3n and 3GC was used to control G-protein binding capability. The GPR-i3n peptide is specially designed for bovine G-protein selectivity and has been proven to have high affinity to G-proteins [Vahlberg, C.; Petoral, R. M., Jr.; Lindell, C.; Broo, K.; Uvdal, K. Langmuir 2006, 22 (17), 7260-7264]. Pure 3GC monolayers show very low protein adsorption capability. In this study, 3GC is chosen as a coadsorbent, with the aim to induce molecular conformational changes during monolayer formation to enhance G-protein adsorption. A full characterization of the mixed monolayers was done. The monolayer thickness and the mass-related surface coverage for both GPR-i3n and 3GC were investigated using radio labeling. The GPR-i3n was labeled by 125I-targeting tyrosine, and the activity was measured by using radioimmunoassay (RIA). The formation and chemical composition of GPRi3n and 3GC monolayers were investigated using X-ray photoelectron spectroscopy, and it is shown that both GPR-i3n and 3GC bind chemically to the gold surface. The interaction between the mixed monolayers and G-proteins was investigated by means of real-time surface plasmon resonance. There is a higher protein binding capacity to the monolayer when the GPR-i3n peptide is intermixed with the 3GC coadsorbent, despite the fact that the 3GC itself has a very low G-protein binding capability. This supports a molecular reorientation at the surface, while 3GC is intermixed with GPR-i3n.

1. Introduction Organic molecular adsorbates can be used to drastically change the physical, chemical and biological properties of a surface. Long-tailed thiols and disulfides have been shown to form selfassembled monolayers (SAMs) on gold, silver, and copper.2 SAMs are well organized and are often referred to as twodimensional crystals. The functionality, specificity, and mobility of these SAMs can be controlled since the tail group of alkane thiol chains can be varied in a wide range. Hydrophobic surfaces,3-5 continuous gradient in hydrophobicity,6 and more flexible surface can be obtained.5 It can be mentioned that thiol chemistry is an important component of the biochip in the Biacore instrument.7 Molecular architecture using thiol chemistry is a * Corresponding author. Mailing address: Kajsa Uvdal, Prof. Sensor Science and Molecular Physics, IFM, Linko¨ping University, S-581 83 Linko¨ping, Sweden. Phone: +46 (0)13 28 1208. Fax: +46 (0)13 28 8969. E-mail: [email protected].

(1) Vahlberg, C.; Petoral, R. M., Jr.; Lindell, C.; Broo, K.; Uvdal, K. R2AAdrenergic receptor derived peptide adsorbates: A G-protein interaction study. Langmuir 2006, 22 (17), 7260-7264. (2) Nuzzo, R. G.; Fusco, F. A.; Allara, D. L. Spontaneously organized molecular assemblies. 3. Preparation and properties of solution adsorbed monolayers of organic disulfides on gold surfaces. J. Am. Chem. Soc. 1987, 109 (8), 2358-2368. (3) Bain, C. D.; Troughton, E. B.; Tao, Y. T.; Evall, J.; Whitesides, G. M.; Nuzzo, R. G. Formation of monolayer films by the spontaneous assembly of organic thiols from solution onto gold. J. Am. Chem. Soc. 1989, 111 (1), 321335. (4) Bain, C. D.; Evall, J.; Whitesides, G. M. Formation of monolayers by the coadsorption of thiols on gold: Variation in the head group, tail group, and solvent. J. Am. Chem. Soc. 1989, 111 (18), 7155-7164. (5) Bertilsson, L.; Liedberg, B. Infrared study of thiol monolayer assemblies on gold: Preparation, characterization, and functionalization of mixed monolayers. Langmuir 1993, 9 (1), 141-149. (6) Liedberg, B.; Tengvall, P. Molecular Gradients of ω-Substituted alkanethiols on gold: Preparation and characterization. Langmuir 1995, 11 (10), 3821-3827. (7) Liedberg, B. K. J. In Affinity Biosensors: Techniques and Protocols; Rogers, K. R., Mulchandani, A., Eds; Methods in Biotechnology Series; Humana Press: Totowa, NJ, 1998.

very promising tool for future biological interaction studies. Surface immobilized peptides and neurotransmitter-based molecules have also been used for surface functionalization,8 and these can be used as powerful tools for investigating specific issues regarding receptor/protein systems.9,10 Such peptidemodified surfaces may be used for drug screening, as sensing layers, and as detecting elements in biosensors.8,11 The seven-helix transmembrane G-protein-coupled receptors (GPCRs) are included in one of the largest families of membranebound receptors. GPCRs are involved in whole series of processes in our body, such as the growth of tissues, metabolic processes within cells, and the synthesis and secretion of proteins. Because of their functional importance, considerable efforts have been made to solve the structure of GPCRs and to understand the mechanism of activation and the location of binding sites for G-proteins.12-14 GPCRs are highly interesting for pharmaceutical research, being a potential drug target.9 (8) Holler, C.; Freissmuth, M.; Nanoff, C. G-Proteins as drug targets. Cell. Mol. Life Sci. 1999, 55 (2), 257-270. (9) Kamel, R.; Eftekhari, P.; Garcia, S.; Berthouze, M.; Berque-Bestel, I.; Peter, J. C.; Lezoualc’h, F.; Hoebeke, J. A high-affinity monoclonal antibody with functional activity against the 5-hydroxytryptaminergic (5-HT4) receptor. Biochem. Pharmacol. 2005, 70 (7), 1009-1018. (10) Morohashi, K.; Yoshino, A.; Yoshimori, A.; Saito, S.; Tanuma, S.; Sakaguchi, K.; Sugawara, F. Identification of a drug target motif: An anti-tumor drug NK109 interacts with a PNxxxxP. Biochem. Pharmacol. 2005, 70 (1), 3746. (11) Norton, W. T. Basic Neurochemistry, 3rd ed.; Siegel, G. J., Albers, R. W., Agranoff, B. W., Katzman, R., Eds.; Little, Brown & Co.: Boston, 1981. (12) Petoral, R. M., Jr.; Wermelin, K.; Dahlstedt, E.; Hellberg, J.; Uvdal, K. Adsorption of n-butyl-substituted tetrathiafulvalene dodecanethiol on gold. J. Colloid Interface Sci. 2005, 287 (2), 388-393. (13) Schoneberg, T.; Schultz, G.; Gudermann, T. Structural basis of G proteincoupled receptor function. Mol. Cell. Endocrinol. 1999, 151 (1-2), 181-193. (14) Chow, E.; Wong, E. L. S.; Bocking, T.; Nguyen, Q. T.; Hibbert, D. B.; Gooding, J. J. Analytical performance and characterization of MPA-Gly-Gly-His modified sensors. Sens. Actuators, B: Chem. 2005, 111, 540-548.

10.1021/la063447f CCC: $37.00 © 2007 American Chemical Society Published on Web 07/10/2007

Mixed Monolayers to Promote G-Protein Adsorption

Langmuir, Vol. 23, No. 16, 2007 8475

Figure 2. The structure of 3GC.

Figure 1. Schematic structure of a GPCR spanning its membrane.

The seven transmembrane domains are connected by three extra- and three intracellular alternating peptide loops (see Figure 1). Binding of a ligand to the extracellular parts of the receptor induces conformational changes in the receptor, which enables the interaction between the intracellular parts of the receptor and the G-protein. It has earlier been shown that the second and third intracellular loops and the C-terminal tail of an R-adrenergic receptor are important sites for G-protein binding and interactions.13,15 We have previously shown that the orientation of the short arginine-linked-molecules ArgCys and Arg-cysteamine will influence the interaction with the G-protein.16,17 The importance of arginine as a building block of the C-terminal of the third intracellular loop has been investigated.18 One peptide, GPRi3c, mimicking this third intracellular loop, was synthesized and compared to two analogues. An exchange of the arginine residues to another positively charged amino acid residue (i.e., lysine) reduced the interaction with the G-protein. Exchanging arginine residues to a neutral charged alanine, G-protein interaction is reduced considerably. In a recent work, the G-protein interaction of three different peptides, GPR-i2c, GPR-i3n, and GPR-i3c was reported.1 The names of GPR-i3n and GPR-i3c derived from the fact that the peptides mimic the N-terminal of the third intracellular loop and the C-terminal of the third intracellular loop of an R2-adrenergic receptor, respectively. It is shown that the G-protein keeps its native conformation during the interaction with GPR-i3c. It should be noted that the choice of buffer is of main importance for keeping G-protein functionality under physiological conditions.17,16 Since synergy effects between different parts of the intracellular loops with respect to both coupling and activation are expected, it is of main interest to study each of these peptides separately and in cooperation.19-21 (15) Eason, M. G.; Liggett, S. B. Identification of a G(S) coupling domain in the amino-terminus of the 3rd intracellular loop of the R2a-adrenergic receptor: Evidence for distinct structural determinants that confer G(S) versus G(I) coupling. J. Biol. Chem. 1995, 270 (42), 24753-24760. (16) Petoral, R. M.; Uvdal, K. Arg-Cys and Arg-cysteamine adsorbed on gold and the G-protein-adsorbate interaction. Colloids Surf., B: Biointerfaces 2002, 25 (4), 335-346. (17) Uvdal, K.; Vikinge, T. P. Chemisorption of the dipeptide Arg-Cys on a gold surface and the selectivity of G-protein adsorption. Langmuir 2001, 17 (6), 2008-2012. (18) Petoral, R. M.; Herland, A.; Broo, K.; Uvdal, K. G-protein interactions with receptor-derived peptides chemisorbed on gold. Langmuir 2003, 19 (24), 10304-10309. (19) Eason, M. G.; Liggett, S. B.; Chimeric mutagenesis of putative G-protein coupling domains of the R2A-adrenergic receptor. Localization of two redundant and fully competent G(i) coupling domains. J. Biol. Chem. 1996, 271 (22), 1282612832.

In this study, mixed monolayers of the positively charged peptide, GPR-i3n, and the shorter, inactive N-formyl-(Gly)3(Cys) (called 3GC) were investigated. Both GPR-i3n and 3GC are linked to the gold surface through thiol coupling chemistry. The peptide design, in combination with the choice of a short inactive 3GC as a coadsorbent, at different mixing ratios, is aimed to control the GPR-i3n molecular orientation at the surface, to further promote G-protein binding/interaction. 2. Experimental Details 2.1. Syntheses and Purification of Peptides. One of the peptides used in this study, GPR-i3n, consists of 13 amino acids and is derived from the third intracellular loop of the R2-adrenergic receptor (Figure 1). Three glycines are added as spacer groups. A cysteine is used as the anchoring group for thiol coupling chemistry of the peptide to the gold surface. The second peptide, 3GC, has the same anchoring and spacer groups as GPR-i3n and is terminated with an N-formyl group. Solid-phase peptide synthesis (SPPS) was used to synthesize the GPR-i3n by standard Fmoc chemistry, utilizing a Pioneer peptide synthesizer (Applied Biosystems). Trifluoroacetic acid (TFA) was used to cleave, the peptide and the protecting groups from the resin. The purification was carried out with HPLC reverse-phase chromatography with a C-8 preparative column, using a ProStar 230 delivery system and a ProStar 330 photodiode detector (Varian). The mobile phase consisted of 0.1% TFA in Milli-Q water and acetonitrile, and the flow rate was set to 10 mL/min with a pressure between 150 and 200 atm. Finally, the purity of the obtained peptide was verified by matrix-assisted laser desorption ionization timeof-flight mass spectrometry (MALDI-TOF MS). The GPR-i3n peptide was, after purification, lyophilized and stored at -70 °C. The shorter 3GC peptide (Figure 2) was also synthesized, via the SPPS described above. 2.2. Sample Preparation. Pure polycrystalline gold surfaces, with a preferential (111) surface, were prepared through the following procedure: Silicon surfaces were cleaned by methods known as TL-1, which consists of 28% H2O2/25% NH3/H2O, 1:1:5 (v/v), and TL-2, which consists of 28% H2O2/37% HCl/H2O, 1:1:6, (v/v).22 The samples were, immediately after the cleaning procedure, mounted in a Balzers UMS 500P electron beam evaporation system. The samples were precoated with a Ti layer with a thickness of 20-25 Å, at an evaporation rate of 2 Å s-1 followed by the evaporation of the 2000 Å thick Au layer at a rate below 5 Å s-1. The base pressure was 5 × 10-9 Torr, and the pressure during the evaporation was 2 × 10-8 Torr. (20) Wade, S. M.; Lim, W. K.; Lan, K. L.; Chung, D. A.; Nanamori, M.; Neubig, R. R. G(i) activator region of R2A-adrenergic receptors: Distinct basic residues mediate G(i) versus G(s) activation. Mol. Pharmacol. 1999, 56 (5), 1005-1013. (21) Wade, S. M.; Scribner, M. K.; Dalman, H. M.; Taylor, J. M.; Neubig, R. R. Structural requirements for G(o) activation by receptor-derived peptides: activation and modulation domains of the R2-adrenergic receptor i3c region. Mol. Pharmacol. 1996, 50 (2), 351-358. (22) Kern W, P. D. Cleaning solutions based on hydrogen peroxide for use in silicon conductor technology. RCA ReV. 1970, 31.

8476 Langmuir, Vol. 23, No. 16, 2007 The gold surfaces were cleaned with the TL-1 procedure and thoroughly rinsed with Milli-Q water just before being immersed into the molecular solution. The adsorption was done at room temperature for more than 48 h, and the beakers were kept in darkness to minimize light-initiated reactions. The gold surfaces were, after molecular adsorption, moved to a beaker with pure water (Milli-Q), ultrasonicated for 5-10 min in order to dissolve any loosely bound, physisorbed molecules, and then carefully rinsed/flushed with water before being gently dried under a gas flow of N2. Gold samples supplied from Biacore AB were used for the radioimmunoassay (RIA). These samples were polycrystalline gold films with a thickness of about 500 Å on a glass support. Prior to use, these surfaces were also cleaned using the TL-1 procedure, described above. The peptide solutions based on degassed water were prepared by mixing the peptide and 3GC in different molar ratios to a total molecular concentration of 70 µM. 2.3. X-ray Photoelectron Spectroscopy (XPS). The XPS experiments were performed on a VG spectrometer, equipped with a CLAM2 analyzer and a twin Mg/Al anode. The measurements were carried out using unmonochromatized Mg KR photons (1253.6 eV). The analyzer resolution was determined from the full width at half-maximum (fwhm) of the Au (4f7/2) peak, which was 1.3 eV with a pass energy of 50 eV. The power of the X-ray gun was 300 W. The temperature in the analysis chamber was approximately 28 °C, and the pressure was approximately 4 × 10-10 Torr during the measurements. The binding energy scale of the XPS spectra was aligned using the Au (4f7/2) peak positioned at 84.0 eV. The measurements were based upon photoelectrons with a take-off angle of 30° relative to the surface normal of the gold substrate. The software used to analyze the peak positions and calculate the elemental composition was VGX900 data analysis software. 2.4. Null Ellipsometry. The refractive indexes of the pure gold samples were measured, prior to incubation in molecular solution, in order to collect the average values of the refractive index of the substrate. Molecular adsorption was done as described above. Samples with freshly prepared, and mixed monolayers were washed with Milli-Q water and immediately blown dried in nitrogen gas before the thickness of the peptide mixed monolayer was measured. The samples were measured in five spots on about five to seven samples for each modified surface, in air, with an Auto-El III Rudolph Research ellipsometer (USA). Single-wavelengths ellipsometry was performed with a He-Ne laser light source (λ ) 632.8 nm) with an angle of incidence of 70°. We assume that the refractive index of the peptide-based mixed monolayers is n ) 1.46523-25 and the complex refractive index of the ambient is set to 1.00. 2.5. RIA. RIA was used in order to quantify the surface mass density of adsorbed GPR-i3n peptide on the surface. In the present study, GPR-i3n was labeled with 125I (τ1/2 ) 60 days, (Amersham Biosciences, U.K.)) and the total amount of adsorbed labeled peptide was measured from the surface activity. This was recalculated into surface mass density.26 The GPR-i3n was labeled with 125I using an Iodobead iodination kit (Pierce, USA) according to the manufacturer’s instructions. Since the molecular size of GPR-i3n is small, the solution with labeled peptides (without Iodobeads) was not filtrated with a desalting gel column, but was instead dialyzed in pure water using a dialyzing membrane (Spectrapor, USA) with a pore size of 1000 Da. Sample volumes (1 mL) were regularly taken, one per hour, from the dialysate (5000 mL, H2O), and the activity was checked. The dialyzation was done until the activity of the dialysate was less than 5000 counts per minute (CPM). (23) Tengvall, P.; Lundstrom, I.; Liedberg, B. Protein adsorption studies on model organic surfaces: An ellipsometric and infrared spectroscopic approach. Biomaterials 1998, 19 (4-5), 407-422. (24) Tengvall, P.; Askendal, A.; Lundstrom, I. Studies on protein adsorption and activation of complement on hydrated aluminium surfaces in vitro. Biomaterials 1998, 19 (10), 935-940. (25) Elwing, H. Protein absorption and ellipsometry in biomaterial research. Biomaterials 1998, 19 (4-5), 397-406. (26) Guide to Radioiodination Techniques; Amersham Life Science, Ltd.: Buckinghamshire, U.K., 1993.

Balau et al. The solution of labeled GPR-i3n was stored for 1 month during the experiments, at room temperature, protected from light. The purity was checked with MALDI-TOF MS before and after labeling in order to ensure that the labeling and the storage time did not affect the solution quality. The activity of the 125I peptide was measured for 10 min using a gamma counter, reporting activity in CPM. The activity values were correlated with the amount of adsorbed protein. A control experiment with the 125I dialysate was made, showing a very low adsorption of free 125I on the surface. All the surfaces were saved and measured again over 2 days, once per day, to take into account the radioactive half-life of iodine. The XPS results show that the tyrosine residue was diiodinated, but this would not affect the reactivity of the peptide compared with the monoiodinated product because the samples were compared with the diiodinated reference. If the activity per weight unit is known, then the surface mass density can be calculated after measurements of the activity on the sample with known surface areas. 2.6. Surface Plasmon Resonance (SPR) and Protein Preparation. The SPR measurements were performed with a Biacore 2000 instrument (Biacore AB, Sweden) using surfaces with dimensions of 10 × 10 mm and with four flow channels, each measuring an area of ∼0.5 × 2.5 mm. The functional bovine brain G-protein mixture used in the SPR experiments was delivered from Calbiochem. The mixture contains the following heterotrimeric G-protein: GoR (∼4-5 µM), GiR-1 (∼1-2 µM), GiR-2 (∼1-2 µM), and GiR-3 ( 14). For this reason, two clean gold surfaces were incubated: one in 100% GPR-i3n solution followed by 100% 3GC solution, and the second in a 100% 3GC solution followed by incubation in 100% GPR-i3n solution. The ellipsometric thickness of the GPR-i3n monolayer was 8 ( 1 Å and increased

Mixed Monolayers to Promote G-Protein Adsorption

to 17 ( 1 Å after incubation in a 100% 3GC solution, but there was no evidence of increasing the thickness of the 3GC monolayer (almost the same thickness, 12 Å) after incubation in 100% GPRi3n peptide solution. This suggests that the thickness increase upon adding 3GC may correspond to a higher packing density of the shorter peptide, resulting in reorganization of GPR-i3n on the surface. This is in good agreement with the direct measurement of the surface concentration through radio labeling, showing a drastic decrease in GPR-i3n upon adding a small amount of 3GC. 3.2. Protein Adsorption. The protein interaction study was done to evaluate the amount of material immobilized while changing the conformation of the peptide in presence of the coadsorbent 3GC. From the protein interaction study and RIA, we observed that 3GC clearly influences the G-protein binding. The SPR response of G-protein adsorption on a mixed monolayer prepared from a solution of GPR-i3n and 3GC as a function of the surface concentration of GPR-i3n is shown in Figure 6a. Initially, there is an increase in protein adsorption with increasing amount of 3GC at the surface. The maximum response for G-protein is 8.6 ( 0.7 (× 102 RU) when the surface concentration of GPR-i3n is 0.7 ng/mm2. Upon following the concentration gradient even further, there is a drastic decrease in the protein adsorption. For the monolayers with a high concentration of 3GC, almost no protein adsorption occurs. Only for a GPR-i3n surface concentration of 0.6 ng/mm2 is the protein response less than 0.5 (× 102 RU), which indicates a more favorable molecular orientation of GPR-i3n while 3GC is present in low concentrations. Another way to present this is to express the SPR response as a function of the total number of molecules per GPR-i3n (Figure 6b). A decrease in G-protein binding occurs when the relative number of GPR-i3n on the surface is reduced from about 20% (1 GPR-i3n over five total molecules) to about 10% (1 GPR-i3n over 10 total molecules). The maximum G-protein binding is obtained when 20% of the molecules on the surface are GPR-i3n. Our hypothesis is that the 3GC in small concentrations exposes the peptide to interact more efficiently with G-protein. At a ratio of 1:4 in terms of the number of molecules for GPR-i3n and 3GC on the surface, G-protein adsorption is enhanced by 18%

Langmuir, Vol. 23, No. 16, 2007 8479

compared to that on monolayers of GPR-i3n. It is worth mentioning that this value was obtained from the relative surface concentrations by taking into account the molecular weight of the two peptides. The static contact angles are also measured for completion. A higher contact angle is found in the range of GPR-i3n [1-0.70] ng/mm2 (around 40 compared with just 20 in the range of GPRi3n [0.6-0]), indicating that the molecular reorientation and distribution are the main reasons for different G-protein bindings.

Conclusion Pure and mixed monolayers of GPR-i3n and 3GC at different molar ratios were prepared for G-protein adsorption studies. GPRi3n is a peptide mimicking the third intracellular loops of the R2-adrenergic receptor, and 3GC is a shorter inactive peptide used to promote monolayer self-assembly. Both GPR-i3n and 3GC bind chemically to the substrate. The monolayer thicknesses for mixed monolayers were measured, and the mass-related surface coverage was estimated as a function of the molar fraction of the GPR-i3n peptide in solution from which the monolayers were prepared. Monolayer purely based on the inactive molecule 3CG, was a good choice as a reference for G-protein interaction. A mixture of GPR-i3n peptides and the small inactive 3GC molecule on the surface increased the total response of adsorption between 1 and 0.70 ng/mm2 of GPR-i3n on the surface. The optimum mixing ratio of GPR-i3n/3GC is found to be 1:4 for maximum G-protein adsorption. At this ratio, G-protein adsorption is enhanced by 18% compared to monolayers of GPR-i3n. The reason for this is suggested to be related to conformational changes of the GPR-i3n peptide while assembled together with 3GC, in such way that it is more available for G-protein interaction. Acknowledgment. This project was supported by grants from the Swedish Research Council (Vetenskaprådet) and a Marie Curie Early Stage Research Training Fellowship of the European Community’s Sixth Framework Programme under Contract Number MEST-CT-2004-504272. P. Nygren and T. Hellerstedt are acknowledged for synthesizing the 3GC molecule. LA063447F