Gold Nanoparticle-Induced Formation of Artificial Protein Capsids

Letter pubs.acs.org/NanoLett

Gold Nanoparticle-Induced Formation of Artificial Protein Capsids Ali D. Malay,1,2 Jonathan G. Heddle,*,2 Satoshi Tomita,*,1 Kenji Iwasaki,3 Naoyuki Miyazaki,3 Koji Sumitomo,4 Hisao Yanagi,1 Ichiro Yamashita,1 and Yukiharu Uraoka1 1

Graduate School of Materials Science, Nara Institute of Science and Technology (NAIST), and CREST, Japan Science and Technology Agency, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan 2 Heddle Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan 3 Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, and CREST, Japan Science and Technology Agency, 3-2 Suita, Osaka 565-0871, Japan 4 NTT Basic Research Laboratories, 3-1 Morinosato-Wakamiya, Atsugi, Kanagawa 243-0198, Japan S Supporting Information *

ABSTRACT: Gold nanoparticles are generally considered to be biologically inactive. However, in this study we show that the addition of 1.4 nm diameter gold nanoparticle induces the remodeling of the ring-shaped protein TRAP into a hollow, capsid-like configuration. This structural remodeling is dependent upon the presence of cysteine residues on the TRAP surface as well as the specific type of gold nanoparticle. The results reveal an apparent novel catalytic role of gold nanoparticles. KEYWORDS: Gold nanoparticles, artificial capsid, protein remodeling, nanospheres

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ollow, polyhedral proteins are commonly found in nature and are of great interest to nanoscience because of their potential to act as cargo carriers and nanoreaction vessels. Designing and building such structures however remains problematic. Here we show that the ring-shaped protein TRAP (trp RNA-binding attenuation protein) can be engineered such that in benign reaction conditions, its normally stable structure can undergo an unusual transformation and form a hollow sphere-like particle reminiscent of a virus capsid. This transition occurs due to a previously unreported role of gold nanoparticles, which appear to catalyze the protein remodeling. The starting material, TRAP, is found in species of Bacillus where its biochemical role is in the feedback control of tryptophan synthesis.1 Under normal conditions, TRAP folds into a thermostable ring composed of 11 identical monomers each with a molecular weight of 8.3 kDa.1 The protein ring offers four distinct and addressable surfaces (the internal and external faces and two “end” faces). Because of its small size and toroidal shape as well as high stability,2 TRAP has been investigated as a possible component of artificial nanostructures3,4 and has been used to deliver gold nanodots to a semiconductor device.4,5 In this study, in order to investigate further the interaction of TRAP with gold nanoparticles we mutated the lysine (Lys) residue at position 35 of the protein to cysteine (Cys), such that each TRAP ring carries 11 identical Cys substitutions around the outer surface (Figure 1). As TRAP contains no native cysteines, these represent the sole Cys residues in the resulting mutant protein, named TRAPK35C. TRAP-K35C offers an ideal opportunity for investigating controlled protein-gold particle interactions because of the strong affinity of cysteine side chains for gold. Details of methods used to construct mutants, express and purify proteins and details of data collection can be found in the Supporting Information. © 2012 American Chemical Society

Figure 1. Location of the K35C mutation in TRAP. Crystal structure of TRAP (PDB 1QAW16) shown along (a) the 11-fold rotational axis (“top view”) and (b) orthogonal to it (“side view”) showing the location of the K35C mutation. The substituted Cys residues are displayed as space-filling atoms.

Purified TRAP-WT and TRAP-K35C appeared identical under transmission electron microscopy (TEM) with both proteins clearly forming toroids (Figure 2a,b). When TRAPWT was incubated with 1.4 nm diameter gold nanoclusters Received: January 18, 2012 Revised: February 10, 2012 Published: March 13, 2012 2056

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concentration of NG (15 μM), TEM revealed a single CLS species with a diameter of approximately 21−22 nm (Figure 3a), whereas upon incubation with the higher concentrations of NG, an increasingly higher proportion of the smaller, 15−16 nm diameter CLS were observed (Figures 3b−d). This likely reflects the formation of two alternative, ordered arrangements of different numbers of protein subunits. The fact that the higher concentrations of NG tend to favor the formation of the smaller species of CLS may be due to the higher probability of catalytic coupling of subunits via NG. CLS formation readily occurred at pH values above 7 (Supporting Information Figure S2e,f). At pH 5−6, CLS failed to form; instead opaque structures approximately 10 nm in diameter are observed, presumably denatured TRAP-K35C (Supporting Information Figure S2a,b). Many of these structures are decorated on the outer surface with NG. At pH 7, a mixture of heterogeneous material decorated with NG as well as CLS not associated with NG were observed (Supporting Information Figure S2c,d). To determine the overall shape of the CLS, cryo-electron tomography (cryo-ET) as well as tapping-mode atomic force microscopy (AFM) were carried out (Figure 4). For cryo-ET (Figure 4a−c), a sample mixture containing TRAP-K35C plus NG was suspended in vitreous ice, and a series of images (single-axis tilt series) of five representative particles were recorded. The reconstructed images (Figure 4b) reveal that the particles have a hollow shell structure with a roughly spherical shape. AFM imaging (Figure 4d,e) was used to measure the height of the CLS, which was found to be approximately 20 nm. The combination of cryo-ET and AFM analyses allow us to conclude unambiguously that the CLS are hollow, spherical protein shells. The exact mechanism of the gold interaction with TRAPK35C remains unknown. In some cases TEM observations showed that the NG particles were not visibly associated with the CLS while in other cases they appeared to be attached to the protein (see Figures 2−4, Supporting Information Figure S3). To investigate this further, the products of the TRAPK35C plus NG mixture were fractionated by sucrose density sedimentation. Spectrophotometric and protein gel analyses showed that for a reaction mixture containing 60 μM each of

Figure 2. Addition of NG induces the reassembly of TRAP-K35C into CLS. Samples were visualized using TEM (scale bars = 40 nm). Protein concentration was 0.5 mg mL−1. (a) Purified wild-type TRAP. (b) Purified TRAP-K35C (inset, region enlarged 3.5× showing regular TRAP ring structures). (c) wild-type TRAP incubated with 120 μM NG. (d) TRAP-K35C incubated with 120 μM NG.

(nanogold; NG), the ring shapes were largely conserved (Figure 2c). In contrast, incubation of TRAP-K35C with the same NG induced dramatic changes in appearance, showing numerous large capsid-like structures (CLS) over the entire TEM grid (Figure 2d). The CLS appear to have an electron dense outer perimeter and a less dense center. The CLS partition into two discrete sizes, a smaller species of approximately 15−16 nm in diameter, and a larger species of approximately 21−22 nm in diameter. Control experiments show that CLS formation depends on the location of the mutated cysteine on the TRAP structure as well as on the size of the gold cluster and the nature of the outer protecting ligand (Supporting Information Figure S1). Intriguingly, the distribution ratio between the two species of CLS could be modulated by changing the concentration of NG in the reaction within the range of 15−120 μM. At the lower

Figure 3. The size distribution of CLS varies with NG concentration. Protein concentration was 0.5 mg mL−1 (60 μM) throughout. The top panel shows TEM images (scale bars = 40 nm) and the bottom panel shows the size distribution based on measuring the diameters of 500 particles per sample. (a) TRAP-K35C with 15 μM NG. (b) TRAP-K35C with 30 μM NG. (c) TRAP-K35C with 60 μM NG. (d) TRAP-K35C with 120 μM NG. 2057

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therefore that NG may act as a catalyst for CLS formation. How catalysis proceeds is not yet clear; however, some rearrangement of the TRAP protein structure must take place to allow it to form CLS. It seems likely that this rearrangement is via binding of TRAP (through cysteines) to the surface of the NG. Time course experiments using TEM revealed that NG can induce the assembly of the artificial capsids in as little as 1 min and proceeds essentially to completion within 1 h (Figure 5). The results also suggest that TRAP-K35C proceeds to the formation of the CLS via an unstructured intermediate, which possibly consists of unfolded or partially unfolded TRAP-K35C subunits. The present study is novel in two main aspects. First of all this is to our knowledge the first report of gold clusters inducing a dramatic reorganization of protein architecture. While the catalytic activity of certain types of gold clusters has been well established,6,7 these have been confined to reactions involving small molecules. Protein structural changes brought about by interactions with nanoparticles typically involve the loss of structure or function brought about by protein denaturation8−10 or the formation of a protein “corona” surrounding the nanoparticles11−13 rather than formation of higher order structures. In another recent report, gold nanoparticles were able to induce tessellation of icosahedral virus capsids to form spherical structures with a range of diameters.14 The exact role of the nanogold cluster in CLS formation in the present system remains obscure and will be the subject of future study. We have tested a number of gold nanoparticles of different sizes and/or outer ligands for their ability to promote CLS formation and have found that efficient and complete formation of CLS is only seen for 1.4 nm diameter Au-NP coated with a tris (aryl) phosphine-based ligand. These results suggest that a particular cluster geometry and the electrostatic charge of the ligand play an important role in the CLS formation reaction. Second, results are significant in demonstrating that a stable, ring-shaped protein oligomer can undergo rapid reorganization into hollow spherical shells having fixed dimensions. The existence of two discrete sizes of CLS strongly suggest the formation of ordered protein superstructures as opposed to disordered aggregates. The similarity in appearance to icosahedral viral capsids is striking; it is interesting to note that some viral capsid proteins, which normally form icosahedral shells, can be induced to form alternative structures such as 2D arrays or tubes with modest changes to salt concentrations or pH.15 Further studies to elucidate the highresolution structure of the CLSs are ongoing.

Figure 4. TRAP-K35C CLS are hollow, spherical shells. (a−c) CryoEM and tomographic reconstruction of TRAP-K35C CLS. Scale bars = 40 nm. (a) Cryo-electron micrography (cryo-EM) image of the sample. (b) Isosurface views of five representative particles extracted from a reconstructed volume. The views of the bottom row were generated by 90° rotation of the upper particles. (c) The central sections of tomographic subvolumes that include one particle each, extracted from the total reconstructed volume. Particles are the same as in (b) and are shown parallel (top row) and perpendicular (bottom row) to the electron beam direction. Crosshairs seen in the center of each particle are added as part of the image processing procedure. The central sections of the subvolumes parallel to the electron beam (top row) show weak density at the top and bottom of the particles. This is likely an artifact caused by the missing wedge of density expected with single-axis tomography and accounts for the “holes” in the structures seen in (b). (d,e) Tapping-mode AFM under aqueous buffer. The line across the sample in panel d is shown as a height profile in panel e. The image shows a sphere-like particle with a height of around 20 nm. The diameter of the particle is also likely close to 20 nm after taking into account the tip radius (approximately 8 nm) and sample drift due to poor adhesion to the mica surface.

protein monomer and NG, the majority of the NG did not fractionate with the CLS fraction (results not shown). Taken together with the TEM results, these results suggest that the NG, while required for CLS formation, are not a necessary part of the final structure and appear there only when stochastically captured during the CLS assembly process. We propose

Figure 5. Time course of CLS formation. The reaction contained 0.5 mg mL−1 TRAP-K35C (60 μM) and 60 μM NG. Samples (4 μL) were immediately applied to TEM grids after the specified times: (a) 1 min incubation; (b) 6 min incubation; and (c) 1 h incubation. Scale bars = 100 nm. At the earlier time points, a large amount of apparently unfolded protein is visible. 2058

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(13) Rocker, C.; Potzl, M.; Zhang, F.; Parak, W. J.; Nienhaus, G. U. Nat. Nanotechnol. 2009, 4 (9), 577−580. (14) Aljabali, A. A. A.; Barclay, J. E.; Cespedes, O.; Rashid, A.; Staniland, S. S.; Lomonossoff, G. P.; Evans, D. J. Adv. Funct. Mater. 2011, 21 (21), 4137−4142. (15) Lavelle, L.; Gingery, M.; Phillips, M.; Gelbart, W. M.; Knobler, C. M.; Cadena-Nava, R. D.; Vega-Acosta, J. R.; Pinedo-Torres, L. A.; Ruiz-Garcia, J. J. Phys. Chem. B 2009, 113 (12), 3813−3819. (16) Chen, X.; Antson, A. A.; Yang, M.; Li, P.; Baumann, C.; Dodson, E. J.; Dodson, G. G.; Gollnick, P. J. Mol. Biol. 1999, 289, 1003−1016.

In summary, we have demonstrated a dramatic, ordered remodeling of protein structure that is induced by gold nanoparticles. TRAP-K35C, upon mixing with specific gold nanoparticles, is able to form hollow sphere-like structures that appear similar to virus capsids. There are no reports of TRAP protein having a close relationship to viral protein sequences, implying that that the method used to form the artificial capsids may be widely applicable and could lead to the use of gold nanoparticles in the facile design of a range of tailor-made protein cargo-carriers.

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ASSOCIATED CONTENT

S Supporting Information *

Additonal information and figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: (J.G.H.) [email protected]; (S.T.) [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for technical assistance from S. Fujita, M. Fujihara, K. Miyake, and Y. Kawai with TEM observation. The authors thank Jeremy Tame for critical reading of the manuscript and Takayuki Kato and Keiichi Namba of the Graduate School of Frontier Bioscience, Osaka University, for kindly providing the cryo-electron microscope facilities. J.G.H. was funded by RIKEN. A part of this work was supported by Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Agency (JST) and Grant-in-Aid for Scientific Research (B) (no.22310079) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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