Oriented Protein Adsorption to Gold Nanoparticles ... - ACS Publications

Nov 29, 2010 - Chemistry Department, Georgetown University, 37th and O Streets NW, ... of proteins to gold nanoparticles (Au NPs) through the tetracys...
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Oriented Protein Adsorption to Gold Nanoparticles through a Genetically Encodable Binding Motif Alison M. W. Reed and Steven J. Metallo* Chemistry Department, Georgetown University, 37th and O Streets NW, Washington, D.C. 20057, United States Received September 2, 2010. Revised Manuscript Received November 3, 2010 Simple, stable, and specific methods for immobilizing proteins on gold surfaces are needed for the development of applications that rely on the oriented attachment of proteins to gold surfaces. We report a direct, stable, genetically encodable method for the oriented chemisorption of proteins to gold nanoparticles (Au NPs) through the tetracysteine motif (C-C-P-GC-C) while simultaneously suppressing protein physisorption. Mutants of ubiquitin (Ub) and enhanced green fluorescent protein (eGFP) containing the tetracysteine motif were produced and displayed stronger adsorption to the NPs than did native proteins. An eGFP mutant with a dicysteine motif (G-C-C) did not show a significant improvement in binding to Au NPs compared to that of the wild-type protein. The binding of the proteins to Au NPs of various sizes (14, 18, 28, and 39 nm) was explored. The small Ub tetracysteine mutant stabilized several sizes of Au NPs, and the eGFP tetracysteine mutant clearly had the strongest chemisorption to the 18 nm NPs. The control of binding orientation for proteins bearing a tetracysteine motif was demonstrated through the enhanced specific binding of protein-NP conjugates to immobilized targets.

Introduction Throughout the past decade, there has been increasing interest in using nanotechnology to solve problems in biotechnology and medicine.1-5 Consequently, there has been a greater focus on the miniaturization of techniques including biomarker detection methods, the patterning of proteins, and the immobilization of proteins on biosensors.1-3,6 Immobilizing enzymes on gold surfaces has been a key area of focus because of its potential applications in biotechnology processes.6-10 Proteins are attached to surfaces by covalent attachment (chemisorption) or physical adsorption (physisorption). Physisorption does not allow control of the protein’s orientation on the surface, resulting in randomly oriented proteins with potentially perturbed tertiary structures and limited protein functionality.11-13 Proteins such as protein A and antiepidermal growth factor have been physisorbed to Au NPs by exposing the particles to proteins after particle synthesis.11,13 Proteins can also physisorb during NP synthesis, where they act as capping agents that control the size of the NPs.14,15 Genetic engineering methods have also been explored *To whom correspondence should be addressed. Phone: 202-687-2065. Fax: 202-687-6209. E-mail: [email protected]. (1) Christman, K. L.; Enriquez-Rios, V. D.; Maynard, H. D. Soft Matter 2006, 2, 928–939. (2) Cass, A. E. G. Electron. Lett. 2007, 43, 903–905. (3) Mendes, P. M.; Yeung, C. L.; Preece, J. A. Nano. Res. Lett. 2007, 2, 373–384. (4) Bajaj, A.; Rana, S.; Miranda, O. R.; Yawe, J. C.; Jerry, D. J.; Bunz, U. H. F.; Rotello, V. M. Chem. Sci. 2010, 1, 134–138. (5) Agasti, S. S.; Rana, S.; Park, M. H.; Kim, C. K.; You, C. C.; Rotello, V. M. Adv. Drug Delivery Rev. 2010, 62, 316–328. (6) Lee, J. M.; Park, H. K.; Jung, Y.; Kim, J. K.; Jung, S. O.; Chung, B. H. Anal. Chem. 2007, 79, 2680–2687. (7) Cullen, S. P.; Liu, X.; Mandel, I. C.; Himpsel, F. J.; Gopalan, P. Langmuir 2008, 24, 913–920. (8) Staii, C.; Wood, D. W.; Scoles, G. J. Am. Chem. Soc. 2007, 130, 640–646. (9) Baas, T.; Gamble, L.; Hauch, K. D.; Castner, D. G.; Sasaki, T. Langmuir 2002, 18, 4898–4902. (10) Bayraktar, H.; Srivastava, S.; You, C. C.; Rotello, V. M.; Knapp, M. J. Soft Matter 2008, 4, 751–756. (11) El-Sayed, I. H.; Huang, X.; El-Sayed, M. A. Nano Lett. 2005, 5, 829–834. (12) Hu, Y.; Das, A.; Hecht, M. H.; Scoles, G. Langmuir 2005, 21, 9103–9109. (13) Li, T.; Guo, L.; Wang, Z. Anal. Sci. 2008, 24, 907–910. (14) Burt, J. L.; Gutierrez-Wing, C.; Miki-Yoshida, M.; Jose-Yacaman, M. Langmuir 2004, 20, 11778–11783. (15) Mei, B. C.; Oh, E.; Susumu, K.; Farrell, D.; Mountziaris, T. J.; Mattoussi, H. Langmuir 2009, 25, 10604–10611.

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to maintain protein functionality by controlling where the protein is attached to the surface. Repeats of a noncysteine containing 14 amino acid peptide, gold-binding polypeptide 1 (GBP1), have been fused to proteins for surface attachment.16,17 Covalent immobilization methods have typically utilized thiol-terminated linkers for chemisorption to gold surfaces and N-hydroxy-succinimide (NHS) esters for attachment to protein. These methods have been used to immobilize RNase A, RNase S, GTPase, Rab6A, and coenzyme A onto Au NPs.8,9,18,19 In linker methods, the surface of the gold nanoparticle is typically modified by a thiol-terminated linker containing a carboxylate group that is subsequently converted to an NHS ester. The proteins are attached to the NPs through the linkers by amide bonds that form when native lysine residues of the protein react with the NHS ester. This method does not allow for the selection of a specific lysine residue; therefore, each protein may be attached to the NPs through different (or multiple) lysine residues. The lysine residue used to attach the protein may also be required for the proper function of the protein. This method of attachment can often result in proteins with reduced functionality because key residues may not be accessible in proteins that are not oriented on the surface.20-22 Other covalent methods attach proteins directly to the surface via native or engineered cysteine residues and improve the probability of the protein binding in the desired orientation.6,8,9,12,23 Protein G mutants with an N-terminal cysteine residue improved the binding to Au surfaces, but there was no significant improvement in binding as the number of cysteines was increased from 1 to 3.6 (16) Tamerler, C.; Duman, M.; Oren, E. E.; Gungorumus, M.; Xiaorong, X.; Kacar, T.; Parviz, B. A.; Sarikaya, M. Small 2006, 2, 1371–1378. (17) Tamerler, C.; Oren, E. E.; Duman, M.; Veskatasubramanian, E.; Sarikaya, M. Langmuir 2006, 22, 7712–7718. (18) Templeton, A. C.; Chen, S.; Gross, S. M.; Murray, R. W. Langmuir 1999, 15, 66–76. (19) Aubin, M. E.; Morales, D. G.; Hamad-Schifferli, K. Nano Lett. 2005, 5, 519–522. (20) Kim, H.; Kang, D. Y.; Goh, H. J.; Oh, B. K.; Singh, R. P.; Oh, S. M.; Choi, J. W. Ultramicroscopy 2008, 108, 1152–1156. (21) Trzaskowski, B.; Leonarski, F.; Les, A.; Adamowicz, L. Biomacromolecules 2008, 9, 3239–3245. (22) Lousinian, S.; Logothetidis, S. Thin Solid Films 2008, 516, 8002–8008. (23) Andreescu, S.; Luck, L. A. Anal. Biochem. 2008, 375, 282–290.

Published on Web 11/29/2010

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Reed and Metallo All glassware was soaked in a base bath solution (1 L of 95% CH3CH2OH, 120 mL of H2O, and 120 g of KOH) for several days before use. The 14-nm-diameter Au NPs were synthesized in aqueous solution by adding 2 mL of 34 mM trisodium citrate to a stirred, boiling 1 mM HAuCl4 solution (20 mL). The solution was heated for approximately 10 min until it turned bright red. To synthesize the 18-, 28-, and 41-nm-diameter Au NPs, 25 mL of 0.01% HAuCl4 solution was heated with stirring, and 500, 375, and 250 μL of 1% trisodium citrate dihydrate solution were added, respectively. Each solution was heated for approximately 10 min until the color change was complete.31,32 The Au NPs were characterized by UV-vis spectroscopy (Agilent UV-vis spectrometer, model 8453) and analyzed by transmission electron microscopy (JEOL JEM 2100 LaB6 TEM, Supporting Information Figure S7).29 The average diameters of the NPs were 39 ( 12, 28 ( 4, 18 ( 4, and 14 ( 2 nm. The NPs were centrifuged at 14000g for 10 min and resuspended in 10 mM sodium phosphate buffer (pH 8).

Construction and Purification of Ub, eGFP, and Mutants.

Figure 1. Structure of FLAsH (A) and structures and dimensions of ubiquitin (B) and enhanced green fluorescent protein (C).

The tetracysteine motif that we use here (C-C-P-G-C-C) was originally designed by Tsien and co-workers to bind bisarsenical dyes (FLAsH and ReAsH) for in vivo imaging of proteins.24-26 The design components that make this sequence useful for binding fluorescent dyes should also make it useful for binding to Au surfaces. It is small, easily engineered into proteins, and exploits protein structural factors to promote ligand binding, which in this case is the surface of a gold colloid. In the live imaging of cells, FLAsH and ReAsH use two arsenic(III) substituents displayed along one edge of the molecule (Figure 1A) to form covalent bonds to the tetracysteine motif that has been incorporated into a protein.26 The binding of the four cysteine-derived thiols displaces the 1,2ethanedithiol (EDT) from the two As atoms on the fluorophore as the motif forms a tight hairpin around the fluorophore.26,27 The β hairpin promoting PG residues facilitate the positioning of all four cysteine thiols on one face of the peptide.26-28 We reasoned that the design components that facilitated the binding of the four cysteine thiols to As atoms along one face would also enhance the ability of the tetracysteine motif to position multiple cysteine thiols to interact with the surfaces of gold particles, resulting in more stable binding than with simple, unstructured cysteine repeats. The improved binding and control of orientation possible with the tetracysteine motif could be useful in many applications that require stable and oriented attachment of proteins to gold nanoparticles.

Materials and Methods Synthesis of Au NPs. Spherical Au NPs were synthesized by the reduction of HAuCl4 (Strem Chemicals) in the presence of trisodium citrate (Na3C6H5O7.2H2O, Mallinckrodt Chemicals).29,30 (24) Hoffmann, C.; Gaietta, G.; Bunemann, M.; Adams, S. R.; OberdorffMaass, S.; Behr, B.; Vilardaga, J. P.; Tsien, R. Y.; Ellisman, M. H.; Lohse, M. J. Nat. Methods 2005, 2, 171–176. (25) Adams, S. R.; Campbell, R. E.; Gross, L. A.; Martin, B. R.; Walkup, G. K.; Yao, Y.; Llopis, J.; Tsien, R. Y. J. Am. Chem. Soc. 2002, 124, 6063–6076. (26) Madani, F.; Lind, J.; Damberg, P.; Adams, S. R.; Tsien, R. Y.; Graslund, A. O. J. Am. Chem. Soc. 2009, 131, 4613–4615. (27) Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269–272. (28) Martin, B. R.; Giepmans, B. N.; Adams, S. R.; Tsien, R. Y. Nat. Biotechnol. 2005, 23, 1308–1314.

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DNA encoding Ub and eGFP was amplified by the polymerase chain reaction (PCR) and cloned into the pET-151/D-TOPO vector (Invitrogen). Mutagenesis was used (Quikchange II, Stratagene) to generate Ub and eGFP mutants containing a C-terminal tetracysteine (TC) motif. An eGFP mutant containing half of the tetracysteine motif (G-C-C) was also cloned. The coding regions of all cloned proteins were confirmed by dideoxy-DNA sequencing (Genewiz, New Jersey). Plasmids expressing Ub, eGFP, and their tetracysteine or dicysteine mutants were transformed into BL21 (DE3) star chemically competent cells (Invitrogen). Single colonies from agar plates containing 0.1 mg/mL ampicillin (1 AMP) were grown in 5 mL of Luria-Bertani (LB) broth for 16 h at 37 °C with shaking (250 rpm). LB cultures (1 L) containing 1 AMP were inoculated with the 5 mL cultures and grown at 37 °C with shaking. The expression of the target protein was induced by the addition of isopropyl β-D-1thiogalactopyranoside (IPTG) to a final concentration of 1 mM when the optical density at 550 nm was between 0.5 and 1. The cells were harvested 3 h after induction by centrifugation at 5000 rpm in a Sorvall GSA rotor at 4 °C. After the cells were lysed by sonication (Kontes Micro Ultrasonic cell disrupter), the lysate was collected by centrifugation (10000g, 15 min). The 6xHis-tagged proteins were purified by immobilized metal affinity chromatography (IMAC) with a His-select nickel affinity gel column (Sigma) under native conditions. Fractions from the purification were run in a 12% bis-tris SDS-PAGE gel (Invitrogen) (Figures S1-S4). The SDS-PAGE gel samples for tetracysteine and dicysteine mutants contained 10 mM DTT to reduce disulfide formation. The proteins were desalted with 3000 MWCO Amicon Ultra-4 centrifugal filters (Millipore). Conjugation and Electrophoresis. The proteins were conjugated to the Au NPs in the absence or presence of dithiothreitol (DTT). Protein samples containing DTT were incubated for 30 min at room temperature before the addition of NPs. Conjugation reactions contained 20 nM Au NPs and 6 μM protein in a 10 mM sodium phosphate buffer (pH 8) and 10 mM DTT (if present). The proteins were incubated with the NPs for 10 min (short incubation) or 12 h (long incubation) at room temperature. For agarose gel analysis, aliquots were mixed with glycerol (5% final) and then loaded on gels. Gel electrophoresis was conducted in a 0.8% agarose gel in 0.25 TBE buffer (22.5 mM Tris-borate, 1 mM EDTA, pH 8.3). The gels were run at 75 V for 1 h and scanned with a Canon LiDE 80 color image scanner. Fluorescence Measurements. Fluorescence measurements were conducted on a Photon Technology International (PTI) fluorimeter (model QM-2001-4) with excitation at 488 nm and emission at 509 nm. Samples were washed by centrifugation for (29) Keating, C. D.; Musick, M. D.; Keefe, M. H.; Natan, M. J. J. Chem. Educ. 1999, 76, 949–955. (30) Storhoff, J. J.; Elghanian, R.; Mucic, R. C. M. C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959–1964. (31) Frens, G. Nat. Phys. Sci. 1973, 241, 20–22. (32) Chithrani, B. D.; Ghazani, A. A.; Chan, W. C. Nano Lett. 2006, 6, 662–668.

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Figure 3. Stabilization of 18 nm Au NPs by Ub-TC and eGFP-TC mutants. The NPs were conjugated without (-) and with (þ) 10 mM DTT (short incubation). Figure 2. Ubiquitin (Ub), enhanced green fluorescent protein (eGFP), and mutants. The amino acids at the C terminus of each protein are represented by their one-letter codes. The proteins have N-terminal 6xHis tags for IMAC purification.

10 min at 14000g, followed by removal of the supernatant and resuspension in 10 mM pH 8 sodium phosphate buffer. Samples were washed twice before initial fluorescence measurements were made, and then five washes were conducted with measurements after each wash. The concentration of the NPs was monitored via absorbance at 520 nm. All experiments were performed in triplicate. IMAC Experiments. Protein-Au conjugates (100 μL) were added to 40 μL of a 50% suspension in 10 mM sodium phosphate buffer (pH 8) of His-select nickel affinity gel beads (Sigma) that had previously been blocked with 10% BSA in 10 mM sodium phosphate buffer (pH 8). The samples were rotated at room temperature in a vertical rotator (Caframo) for 12 h. The Hisselect beads were pelleted from the supernatant by centrifugation for 1 min at 1000g. The absorbance at 520 nm was monitored to determine the concentration of the NPs in the supernatant. The experiment was performed in triplicate.

Results and Discussion Two proteins of significantly different size and structure, ubiquitin (Ub) and enhanced green fluorescent protein (eGFP), were used to test the ability of the tetracysteine motif (TC)26 to provide a stable, genetically encodable tag for the oriented binding of proteins to metal NPs (Figure 1). Ub is a 76 amino acid protein with a highly ordered, stable native structure.33,34 The 239 amino acid β-barrel protein, eGFP, is intrinsically fluorescent.35,36 Because eGFP has to be folded to maintain its fluorescence, its fluorescence indicates the retention of its structure and function.35 Molecular cloning techniques were used to introduce the tetracysteine motif (C-C-P-G-C-C) at the C terminus of both Ub and eGFP and to create a control protein, eGFPDC, containing only a dicysteine (DC) motif (G-C-C) (Figure 2). The TC motif contains central residues Pro-Gly, which are amino acids that have a high propensity to form β turns. As seen in the recent NMR structure of a TC-containing peptide, the motif allows the four cysteine residues to be oriented on one side of the chain simultaneously.26 The orientation of the motif should therefore be advantageous over simply attaching a linear chain of cysteine residues to the protein. It was previously shown that (33) Jackson, S. E. Org. Biomol. Chem. 2006, 4, 1845–1853. (34) Vijay-Kumar, S.; Bugg, C. E.; Wilkinson, K. D.; Cook, W. J. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 3582–3585. (35) Tsien, R. Y. Annu. Rev. Biochem. 1998, 67, 509–544. (36) Ormo, M.; Cubitt, A. B.; Kallio, K.; Gross, L. A.; Tsien, R. Y.; Remington, S. J. Science 1996, 273, 1392–1395.

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increasing the number of cysteines in a linear chain (from 1 to 3) did not improve the binding to a Au surface and is more likely to form disulfides.6 Proteins nonspecifically physisorb to metal surfaces, forming a randomly oriented layer.20 To enhance robust and oriented chemisorption through the tetracysteine motif, physisorption of the proteins had to be suppressed. High concentrations of nonionic detergents (Nonidet P-40 and Triton X-114) did not suppress the physisorption of proteins onto the surface of the NPs (data not shown). The small thiol-containing molecule dithiothreitol (DTT) was found to be effective at suppressing the physisorption of proteins to NPs. DTT alone stabilized the particles from aggregation and presumably works by binding the Au surface through its thiol groups that would orient its hydroxyl groups toward the solution, inhibiting the physisorption of proteins and stabilizing the colloids. DTT was also useful in ensuring that the TC motif remained reduced. The native and mutant proteins are both capable of stabilizing the Au NPs against aggregation. The native proteins stabilize the NPs by physisorption, and the mutant proteins stabilize the NPs through chemisorption. Native gel electrophoresis was used to monitor the binding of the proteins to the Au NPs and to distinguish chemisorbed proteins from physisorbed proteins (Figure 3). Proteins Ub, Ub-TC, eGFP, and eGFP-TC are negatively charged (calculated isoelectric points of 5.76, 6.79, 5.58, and 5.58, respectively) and move toward the positive electrode in the gels. Au NPs that are not conjugated to proteins form black aggregates in the wells of the gels and are not mobile because they are not stable in the high ionic strength of the gel buffer. Almost all biochemical systems use buffers that would cause the aggregation of gold NPs in the absence of additional stabilization. The NPs stabilized by proteins form discrete, mobile red bands in the gel. Au NPs stabilized by DTT become purple in the gel buffer; they are not highly charged and have limited mobility in the gels. Conjugation to protein is critical to the mobility of the NPs; therefore, gel electrophoresis provides a simple and effective method for monitoring conjugation.37,38 In the absence of DTT, the 18 nm Au NPs exposed to Ub and Ub-TC formed mobile red bands in the gel. In the presence of DTT, Ub-TC conjugates did not show signs of aggregation and were seen as mobile red bands, and Ub-Au NPs formed immobile purple aggregates (Figure 3). The results indicated that only UbTC was chemisorbed to the 18 nm Au NPs whereas Ub was more weakly adsorbed and removed from the surface via competition with DTT. Longer incubations with DTT yielded similar results (37) Zheng, M.; Huang, X. J. Am. Chem. Soc. 2004, 126, 12047–12054. (38) Zheng, M.; Davidson, F.; Huang, X. J. Am. Chem. Soc. 2003, 125, 7790– 7791.

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Figure 4. Corrected fluorescence intensity of eGFP-TC (0), eGFP-DC (Δ), and eGFP (O) on 18 nm Au NPs after 5 washes. The fluorescence intensity was corrected for the concentration of NPs by dividing by the nM NP concentration. Each experiment was performed in triplicate, and the standard errors are displayed on the graph. The relative corrected fluorescence values of eGFPTC, eGFP-DC, and eGFP at 508 nm were 11.7, 2.1, and 1, respectively.

(Supporting Information Figure S5). If Ub-TC is chemisorbed, it follows that it is oriented with the tetracysteine motif attached to the surfaces of the NPs. In the absence of DTT, both eGFP and eGFP-TC bound and stabilized the 18 nm NPs. In the presence of DTT, eGFP was removed from the surfaces of the NPs but eGFPTC remained conjugated to the NPs (Figure 3), again indicating that the TC-containing mutant was chemisorbed instead of simply physisorbed. Fluorescence measurements were conducted to quantify the ability of the TC motif to enhance the binding of eGFP on the surfaces of the NPs. The intrinsic fluorescence of eGFP was utilized to monitor the levels of protein conjugated to the NPs. It was hypothesized that the chemisorption possible through the TC motif would significantly improve the binding of eGFP-TC to Au NPs as compared to the binding of the native eGFP even in the absence of the suppression of physisorption by DTT. In these experiments, DTT was not used because the eGFP and eGFP-DC samples incubated with DTT aggregated after they were washed by centrifugation and hence could not be compared to eGFP-TC. We further hypothesized that the entire TC motif was required to significantly improve the binding of eGFP to the NPs. The mutant eGFP-DC that contains half of the tetracysteine motif (GCC) was used to test this hypothesis. The Au NPs were incubated with eGFP, eGFP-DC, and eGFP-TC. The fluorescence was monitored as the NPs were washed multiple times to remove unstably bound proteins. After five washes, eGFP-TC on 18 nm Au NPs had 5 times the fluorescence of eGFP-DC and 10 times the fluorescence of eGFP (Figure 4). The substantial retention of the fluorescence of the eGFP-TC-Au NPs indicated that eGFP-TC was more stably adhered to the NPs than either eGFP or eGFP-DC. The eGFP and eGFP-DC were most likely attached to the surfaces of the NPs through physisorption because they were removed by the washes. Because DTT was not added to the samples, it was possible for some eGFP-TC proteins to be physisorbed to the NPs. The significant retention of fluorescence suggests that eGFP-TC was not displaced from the NPs as eGFP and eGFP-DC were; these attached through physisorption. The addition of only two cysteines had little effect on improving the binding, which suggests that the entire TC sequence was necessary for stable chemisorption. The significant retention of fluorescence for the eGFP-TC conjugates even when physisorption of the 18948 DOI: 10.1021/la1035135

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proteins is not inhibited suggests that the protein is binding preferentially through the TC motif to stabilize the NPs. The multiple, oriented cysteine contacts of the TC motif were able to improve the binding with the NPs dramatically and provide significant improvement over physisorption. The effect of the size of the NPs on the binding of proteins was investigated by monitoring the binding of proteins to NPs of decreasing (14 nm diameter) and increasing size (28 and 39 nm diameter). The size of the NPs can influence the binding of proteins to the surface because the increased surface curvature of smaller NPs may decrease the ability of larger proteins to bind to the surface.15,39,40 Gel electrophoresis experiments performed on 14 nm Au NPs revealed that only Ub-TC remained on the NPs in the presence of DTT, indicating the chemisorption of Ub-TC. The native and mutant proteins, eGFP and eGFP-TC, respectively, were displaced from the surface of the NPs by DTT (Figure 5). On larger (28 and 39 nm) Au NPs, Ub remains on the surface in the absence of DTT. In the presence of DTT, no discrete mobile band was observed; however, the smearing indicated that Ub was likely incompletely displaced from the particles by both high (10 and 20 mM, Figure 6a,c) and low (2.5 and 5 mM, Figure S6) DTT concentrations. These results indicate that Ub was subject to displacement by DTT; however, the lack of simple aggregates in the wells indicated that Ub was more difficult to displace from the larger NPs. In the presence of high (10 and 20 mM) and low (2.5 and 5 mM) DTT, Ub-TC remained on the surface of the 28 and 39 nm Au NPs. Thus, Ub-TC stabilized NPs of various sizes (14, 18, 28, and 39 nm) through chemisorption. The discrete red bands in the gel indicate that eGFP and eGFP-TC were not displaced from the surface of the 28 nm NPs in the presence of DTT (Figure 6b,d). The smearing and lack of discrete bands indicate that there was a displacement of eGFP and eGFP-TC from the Au surface as the size of the NPs increased to 39 nm (Figure 6d). These results indicate that neither eGFP nor eGFP-TC provides complete stabilization of the 39 nm NPs in the presence of DTT. The effects of curvature on the surface density of nucleic acids have been studied, and it is known that an increase in curvature results in increased surface coverage.41,42 Although the effects of curvature are more predictable for nucleic acids, these effects are more complex for peptides and proteins. As the NPs decrease in size, the curved surface of the smaller NPs can generally suppress the adsorption of larger proteins.39,40 However, these curvature effects are less predictable than for peptides and nucleic acids because there are more ways for proteins to interact with the surface and also with each other.32,41 The dimensions of eGFP are 4.2  3.2  3.1 nm3, and Ub has dimensions of 2.4  3.5  1.9 nm3; therefore, eGFP is significantly larger than Ub.35,43-45 It is possible that eGFP and eGFP-TC have limited binding to the 14 nm NPs because of the small size and increased curvature of the NPs. This simple model, however, cannot capture all aspects of the protein-particle interaction: although neither eGFP nor eGFP-TC was effectively displaced by DTT from the 28 nm particles, on the largest NPs (39 nm) both eGFP-TC and eGFP were displaced and failed to provide stabilization in the presence (39) Lynch, I.; Dawson, K. A. Nano Today 2008, 3, 40–47. (40) Cedervall, T.; Lynch, I.; Lindman, S.; Berggard, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2050–2055. (41) Cederquist, K. B.; Keating, C. D. ACS Nano 2009, 3, 256–260. (42) Hill, H. D.; Millstone, J. E.; Banholzer, M. J.; Mirkin, C. A. ACS Nano 2009, 3, 418–424. (43) De, M.; Miranda, O. R.; Rana, S.; Rotello, V. M. Chem. Commun. 2009, 2157–2159. (44) PDB ID 1UBQ. Vijay-Kumar, S.; Bugg, C. E.; Cook, W. J. J. Mol. Biol. 1987, 194, 531–544. (45) PDB ID 1C4F. Elsliger, M. A.; Wachter, R. M.; Hanson, G. T.; Kallio, K.; Remington, S. J. Biochemistry 1999, 38, 5296–5253.

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Figure 5. Stabilization of 14 nm Au NPs by proteins. The NPs were run without (-) and with (þ) 10 mM DTT. (A) Short incubation (10 min). (B) Long incubation (12 h).

Figure 6. Protein adsorption onto 28 and 39 nm Au NPs. The physisorption of proteins on 28 nm Au NPs of (A) Ub and Ub-TC and (B) eGFP and eGFP-TC was inhibited with dithiothreitol (DTT). The NPs were run with and without 10 and 20 mM DTT (short incubation). Inhibition of protein binding to 39 nm Au NPs of (C) Ub and Ub-TC and (D) eGFP and eGFP-TC with dithiothreitol (DTT). The NPs were run with and without 10 and 20 mM DTT (short incubation).

of DTT. Smaller globular Ub-TC was capable of consistently stabilizing NPs of various sizes. These experiments show that the size of the NPs has a definite effect on the binding of the proteins. The TC motif is capable of mediating robust binding to the Au NPs in the presence of competing thiol (DTT); however, not all particle sizes will be compatible with all proteins. The preferential orientation of the proteins on the surfaces of the NPs due to the binding of the tetracysteine motif was confirmed by determining the accessibility of the N-terminus of proteins attached to the NPs. The 6xHis tag and the tetracysteine motif are at opposite ends of Ub-TC (N-terminus and C-terminus, respectively). Proteins that are chemisorbed to the NPs through the tetracysteine motif should be oriented with the 6xHis motif away from the NP surface. The increased accessibility of the 6xHis motif for chemisorbed proteins should make it more Langmuir 2010, 26(24), 18945–18950

available to interact with specific binding partners such as immobilized Ni2þ ions. Thus, we used binding to nickel nitrilotriacetic acid (Ni-NTA) immobilized on agarose beads as a functional test of the ability to orient proteins specifically at the surfaces of the NPs. If the proteins bind in an orientation that allows the 6xHis tag to be available, then the red protein-Au conjugates are captured by the beads, which become red and are removed from the supernatant, which becomes clear. If the 6xHis tag is not available, then the NPs cannot attach to the beads and the supernatant remains red. Ni-NTA beads were blocked with BSA to decrease nonspecific binding and were incubated with Ub NPs or Ub-TC NPs. Without protein, the 18 nm Au NPs were pink and the beads remained clear, indicating that the NPs on their own were not significantly captured by the beads (Figure 7). NPs incubated with bovine DOI: 10.1021/la1035135

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Reed and Metallo Table 1. Percentage of 18 nm Au NPs bound to beads in the Ni-NTA assay. The measurements were made in triplicate and the standard deviation is reported

Figure 7. Nickel nitrilotriacetic acid (Ni-NTA) assay to determine the orientation of the Ub-TC on the Au NP surface. The Au NPs are in the supernatant, and the Ni-NTA beads are at the bottom of the tube. The supernatant becomes colorless and the beads become red if the protein-Au conjugates bind to the beads. The protein-Au conjugates were incubated with or without (() 10 mM DTT.

serum albumin (BSA) were used as a control because BSA strongly stabilizes the NPs. In the presence of DTT, Ub-TC NPs remained red, indicating that they were stabilized by Ub-TC, whereas Ub NPs formed black aggregates, indicating that Ub was displaced from the surfaces of the NPs (as expected from previous experiments). The Ub-TC NPs had significant binding to the beads in the absence and presence of DTT (95% bound), but the Ub NPs had less binding (76% binding without DTT and aggregation with DTT, Table 1). When the small protein, Ub, binds to the NPs, the 6xHis tag may be accessible from several positions of physisorption. In the presence of DTT, the NPs aggregated as Ub was displaced from the surfaces of the NPs, indicating that Ub was mostly physisorbed. These results, which were reproducible, are in agreement with the gel electrophoresis experiment that confirmed that Ub-TC has improved binding over that of native proteins and can stabilize the NPs in the presence of DTT. The results show that the tetracysteine tag improves the affinity for the NP surface and that the orientation of the protein on the NPs can be enhanced with the tetracysteine motif. The TC motif, which provides a stable method for the oriented chemisorption of proteins to Au NPs, has an advantage over other methods of immobilizing proteins because it allows one to select a specific site of attachment. Controlling the exact region in which the protein attaches to a surface aids in preserving the activity of the protein by ensuring that residues required for function are not inactivated by the linkers used for immobilization. In addition to preserving activity, the motif provides control of the protein’s orientation. This feature may be particularly useful in applications that require the accessibility of a specific domain on a protein. If a protein interacts with a binding partner through its (46) Pavlickova, P.; Schneider, E. M.; Hug, H. Clin. Chim. Acta 2004, 343, 17– 35. (47) Jung, Y.; Jeong, J. Y.; Chung, B. H. Analyst 2008, 133, 697–701.

18950 DOI: 10.1021/la1035135

Sample

% Au NPs bound

Au (-) DTT Au (þ) DTT AuBSA (-) DTT AuBSA (þ) DTT AuUb (-) DTT AuUb (þ) DTT Au-Ub-TC (-) DTT Au-Ub-TC (þ) DTT

32 ( 5 aggregated 39 ( 1 26 ( 2 76 ( 2 aggregated 95 ( 1 95 ( 0

N-terminus, then the TC motif could be genetically engineered at the C-terminus to ensure that the N-terminus is available for binding. The ability to manipulate the orientation of a protein would be useful in several assays, including those involving recombinant antibodies that require a specific orientation for binding.46,47

Conclusions The tetracysteine motif combines multiple cysteine contacts with protein structure to provide a robust method for attaching proteins to Au NPs of various sizes. Engineering the position of the tetracysteine motif along with the use of DTT to suppress physisorption provides a genetically encodable method of controlling the orientation of the protein on the Au surface. The multiple cysteine contacts of this motif also allow the protein of interest to bind to the NPs even in the presence of physisorbed proteins. The contacts of the TC motif allow the protein to remain chemisorbed to the surface after multiple washings, whereas physisorbed proteins are removed from the surfaces of the NPs. There is a crucial link between the orientation of proteins and their function when interacting with other species. This motif could increase the activity of proteins in applications such as sensor devices by controlling the orientation of these proteins. The robustness and control provided by the tetracysteine motif should facilitate the creation of multifunctional NPs, that is, NPs with multiple active proteins attached. Acknowledgment. Parental Ub and eGFP plasmids were kindly provided by Prof. David Yang. We thank Prof. Tong and Oksana Zaluzhna for their assistance with TEM imaging. Supporting Information Available: TEM images of particles, a detailed description of protein cloning and purification, and additional agarose gels. This material is available free of charge via the Internet at http://pubs.acs.org.

Langmuir 2010, 26(24), 18945–18950