Janus-like Protein Cages. Spatially Controlled Dual-Functional

NANO LETTERS

Janus-like Protein Cages. Spatially Controlled Dual-Functional Surface Modifications of Protein Cages

2009 Vol. 9, No. 6 2360-2366

Sebyung Kang,†,§ Peter A. Suci,‡,§ Chris C. Broomell,†,§ Kenji Iwahori,| Mime Kobayashi,| Ichiro Yamashita,| Mark Young,*,‡,§ and Trevor Douglas*,†,§ Department of Chemistry & Biochemistry, Department of Plant Sciences, and Center for BioInspired Nanomaterials, Montana State UniVersity, Bozeman, Montana 59717, and Nara Institute of Science and Technology, Takayama-cho, Ikoma, Nara, Japan Received March 20, 2009; Revised Manuscript Received May 1, 2009

ABSTRACT Protein cages have been used both as size-constrained reaction vessels for nanomaterials synthesis and as nanoscale building blocks for higher order nanostructures. We generated Janus-like protein cages, which are dual functionalized with a fluorescent and an affinity label, and demonstrated control over both the stoichiometry and spatial distribution of the functional groups. The capability to toposelectively functionalize protein cages has allowed us to manipulate hierarchical assembly using the layer-by-layer assembly process. Janus-like protein cages expand the toolkit of nanoplatforms that can be used for directed assembly of nanostructured materials.

Protein cages, including ferritins, viral capsids, and heat shock proteins, have been widely used as nanoscale building blocks for fabricating higher order nanostructures, such as ordered planar arrays,1-3 nanowires,4,5 nanoelectronic devices,6-8 and layer-by-layer (LbL) assemblies,9-15 either by using their endogenous self-assembly properties2 or by modifying their surfaces to accomplish their directed assembly.9-16 Genetic and chemical modification of the exterior surface of protein cage architectures allows site specific attachment and presentation of various types of molecules including affinity tags, antibodies, fluorophores, carbohydrates, nucleic acids, and targeting peptides.17-28 Multiligand presentation on a single protein cage surface has been achieved by labeling with two different reagents either simultaneously or sequentially17-26 or by assembly of prefunctionalized subunits in controlled ratios.27,28 While the highly symmetric nature of protein cages is advantageous for the formation of isotropic hierarchal structures, it is necessary to break the functional symmetry of the cage to achieve topological control for applications such as directed molecular recognition, controlled anisotropic hierarchal assembly, and polarized multicomponent presentation.29-32 Due to their high symmetry, however, controlling the spatial * Corresponding authors: Professor Trevor Douglas, phone (406) 9946566, fax (406) 994-5117, e-mail [email protected]; Professor Mark Young, phone (406) 994-5158, fax (406) 994-5117, e-mail [email protected]. † Department of Chemistry & Biochemistry, Montana State University. ‡ Department of Plant Sciences, Montana State University. § Center for BioInspired Nanomaterials, Montana State University. | Nara Institute of Science and Technology. 10.1021/nl9009028 CCC: $40.75 Published on Web 05/14/2009

 2009 American Chemical Society

Figure 1. Surface and ribbon diagram representations of LiDps (PDB 1QGH) looking down the 3-fold symmetry axis (left) and a clipped view showing the interior space of the cage (right). C-termini (red) are indicated.

location of functional groups to achieve toposelectivity presents a formidable challenge. In this study, we have genetically modified the surface of a 12-subunit isotropic hollow protein cage, LiDps (DNA binding protein from starved cells from the Gram-positive bacterium Listeria innocua) (Figure 1), and toposelectively modified its surface with two different functionalities using a masking/unmasking method on solid supports (Scheme 1). LiDps is a member of the ferritin superfamily and prevents oxidative damage to DNA by accumulating iron atoms within its central cavity to produce an iron oxide core similar to that of ferritins.33 LiDps consists of 12 identical 18 kDa subunits that self-assemble into a hollow protein cage having tetrahedral 23 symmetry (Figure 1)33 with an outer diameter of 9 nm and an inner cavity diameter of 5 nm (Figure 1). It has been used as a template for mineralizing metal oxides,

Scheme 1. Toposelective Surface Modification of Symmetric Protein Cages through a Masking/Unmasking Technique on Solid Supports

such as iron34 and cobalt,35 cadmium sulfide,36 and platinum.37 The small number of subunits, high temperature stability,34,35 and intrinsic biomineralizing capability33 of LiDps make it an attractive platform for nanomaterials synthesis. In addition, the defined small cavity size allows synthesis of extremely small nanostructured materials.34-37 To adapt LiDps for selective chemical modifications on the exterior surface, four additional amino acids (KLFC) were added to the C-terminus as described previously28 (Figure 1, red). The chemical reactivity of introduced cysteines of KLFC LiDps was tested with cysteine reactive maleimide reagents, maleimide-PEG2-biotin (MPB) and fluorescein-5maleimide (F5M). Chemically modified KLFC LiDps eluted on size exclusion chromatography (SEC) at the same position as wt LiDps (Figure F1 in Supporting Information) suggesting chemical modifications did not alter the protein architecture. The extent of MPB or F5M modification to each protein cage was evaluated by mass spectrometry as described previously.19,28 Mass analyses revealed that, at saturation levels, 11.6 and 11.5 subunits per cage of KLFC LiDps protein cages (12 subunits total) were chemically modified with MPB and F5M on average, respectively (Figure F2 in Supporting Information) suggesting that all the introduced cysteines are equivalently reactive to maleimide reagents. The masking/unmasking approach of symmetric particles on solid supports has been used previously for fabricating site-specific functionalized Janus particles.30,31 We adapted this approach to generate Janus-like LiDps protein cages using activated thiol beads as the solid support (Scheme 1). We used two different types of activated thiol beads for toposelective modification of KLFC LiDps. The two bead preparations (4B and 6B) have different spacers, glutathione Nano Lett., Vol. 9, No. 6, 2009

(1.3 nm) and 2-hydroxypropanol (0.7 nm), respectively, as well as different active group concentrations (1 and 25 µmol of thiol/mL of drained medium, respectively). To protect one side of the symmetric KLFC LiDps from labeling, cages were incubated with either 4B or 6B beads at 4 °C overnight (Scheme 1A). No detectable KLFC LiDps was observed in the washed fraction after overnight incubation suggesting that all the introduced KLFC LiDps was bound to the activated thiols. The first surface modification of both 4B and 6B bound KLFC LiDps was performed using a 10-fold molar excess of MPB to KLFC LiDps subunit at room temperature for 3 h (Scheme 1B). The MPB-reacted KLFC LiDps were eluted from the beads by reduction with a buffer containing 25 mM DTT (final concentration) (Scheme 1C). Free MPB and released 2-thiopyridine were removed by SEC. On-bead MPB-modified KLFC LiDps eluted at the same position as intact KLFC LiDps suggesting on-bead modifications did not alter the protein cage architecture (Figure F3 in Supporting Information). Free cysteines of subunits previously masked by disulfide formation with the beads were subsequently labeled with F5M in solution at room temperature for 3 h (Scheme 1D). The extent of modifications at each step was evaluated using mass spectrometry (Figure 2). Electrospray ionization (ESI) produces a series of multiply charged ions, and the charges (z) are generally distributed as a continuous series with a Gaussian intensity distribution (..., z - 2, z - 1, z, z + 1, z + 2,...) (Figure 2A,C,D,F, bottom panels). If there are multiple species in a mass spectrum, multiple Gaussian charge distributions can be detected and the molecular masses of each species can be determined from the charges and the observed mass-to-charge (m/z) ratio values (Figure 2A,D). Soft ionization using ESI38 makes it possible to obtain masses 2361

Figure 2. Mass spectra of untreated, on-bead-modified, and dual-modified KLFC LiDps protein cages and cartoon representations of modification distributions on the protein cage surfaces. (A, D) Mass spectra of untreated (bottom), MPB modified on 4B (A) or 6B (D) (middle), and MPB-F5M dual-modified (top) KLFC LiDps protein cage subunits. Charge state distributions for untreated (black dots), MPB-modified (red dots), and F5M-modified (green dots) subunits are marked. Putative modification locations are marked with corresponding colored dots on the cartoons and charged peaks are marked every two peaks (bottom). (B, E) Zoomed-in (m/z ) 1010-1100) mass spectra of untreated (bottom), MPB modified on 4B (B) or 6B (E) (middle), and MPB-F5M dual-modified (top) KLFC LiDps protein cage subunits. Charged peaks (18+) are indicated. (C, F) Mass spectra of untreated (bottom), MPB modified on 4B (C) or 6B (F) (middle), and MPB-F5M dual-modified (top) KLFC LiDps protein cages with charged peaks. Calculated and observed masses are indicated.

of protein components individually as well as measure the intact mass of noncovalently associated macromolecular complexes without disturbing the structure.39,40 While subunit mass analysis of samples at each step provides information about relative populations of differentially modified subunits (or species) in the whole populations (Figure 2A,B,D,E), noncovalent mass analysis allows us to determine the degree of subunit modifications in a single protein cage by the corresponding mass increase (Figure 2C,F). Interestingly, the degree of MPB modification of KLFC LiDps on the 4B beads differed from that on the 6B beads. While half of the subunits were modified with MPB on the 4B beads (Figure 2A,B, middle panels (red dots)), only a quarter of the subunits were modified with MPB on the 6B beads (Figure 2D,E, middle panels (red dots)). These results suggest that only one face (6 subunits) of a protein cage binds to the 4B bead surface, whereas 9 subunits of a protein cage (12 subunits) can bind to the 6B bead surface resulting in toposelective modifications as illustrated in Figure 2. Analyses of noncovalent mass spectra revealed mass increases of 2882 and 1665 Da following MPB treatment on 4B and 6B beads, respectively, which correspond to 5.5 and 3.2 MPBs per cage on average (Figure 2C,F). The narrow peak width of noncovalent mass 2362

spectra implies that all populations of KLFC LiDps were modified to a similar degree (Figure 2). These results suggest that all KLFC LiDps had a similar interaction with the thiol activated beads resulting in toposelective modifications as illustrated in Figure 2. Secondary modification with F5M after releasing MPBmodified KLFC LiDps from the bead was successfully achieved (Figure 2). Subunit mass peaks were shifted with respect to the corresponding unmodified KLFC LiDps subunit peaks to m/z values expected for complete attachment of F5M (Figure 2A,B,D,E, top panels (green dots)). Mass increases (2943 and 4013 Da) were observed by noncovalent mass spectrometry of F5M-treated MPB-modified LiDps released from 4B and 6B beads (Figure 2C,F, top panels). These mass increases translate to 6.8 and 9.2 F5M per cage, agreeing well with the numbers of protected subunits (6.5 and 8.8 subunits, respectively). On-bead and subsequent in-solution modifications of KLFC LiDps were expected to produce dual-functionalized Janus-like protein cages, having both an affinity (biotin) and fluorescent (fluorescein) label. To demonstrate their dual functionality directly, dual-functionalized KLFC LiDps cages Nano Lett., Vol. 9, No. 6, 2009

Figure 3. Epifluorescence microscopy of untreated, MPB, F5M, or MPB-F5M dual-modified KLFC LiDps protein cages bound onto the streptavidin coated plate. (A) Epifluorescence images of the streptavidin coated plate after treatments and extensive washings of untreated, MPB, F5M, or MPB-F5M dual-modified KLFC LiDps protein cages, respectively. (B) Pixel counts of grayed-scale images from three independent measurements.

were bound to a streptavidin-coated plate and the fluorescence was detected using epifluorescence microscopy (Figure 3). Unmodified KLFC LiDps, and KLFC LiDps fully modified with MPB or F5M, were used in parallel as controls. While the controls showed some background scattering, a bright fluorescent image was obtained only from the dual-functionalized KLFC LiDps (Figure 3) suggesting that only the dual-functionalized KLFC LiDps protein cages bind selectively to the streptavidin-coated plate and fluoresce. The extent of differential MPB and F5M labeling determined by mass spectrometry, together with the epifluorescence microscopy data demonstrating the dual functionality of protein cages, suggests strongly that modification of KLFC LiDps bound to the beads was toposelective. We corroborated this interpretation with a functional assay in which the asymmetry of the protein cage was exploited to cap an LbL assembly process. Sequential alternate depositions of MPB-modified KLFC LiDps and streptavidin were monitored in real time using the quartz-crystal microbalance (QCM) measurement. Deposition of molecules on the QCM sensor results in resonance frequency decreases (-∆F) which correspond to the masses of deposited molecules.9,15,41 Wildtype LiDps, the KLFC LiDps genetic construct, and both fully F5M-modified and fully MPB-modified KLFC LiDps bound strongly, with slightly different magnitude, to the gold QCM sensor without any surface treatment (Figure F4 in Supporting Information). Alternate deposition of protein cage and streptavidin resulted in sequential decreases in the resonance frequency (Figure 4A). To achieve saturation of binding for each deposition step, we introduced samples (Figure 4, arrows) and allowed binding for 5 min followed by two additional sequential introductions of samples (Figure Nano Lett., Vol. 9, No. 6, 2009

4, *). After three consecutive additions, unbound or loosely bound materials were removed by flowing buffer over the QCM sensor surface (Figure 4, W). Flowing buffer did not change the resonance frequencies indicating stable LbL formation mediated by the strong biotin-streptavidin interactions (Figure 4). While the first sample introductions of each layer, either MPB KLFC LiDps (down double arrow) or streptavidin (down single arrow), resulted in dramatic decreases of the resonance frequency, subsequent (Figure 4, *) introductions did not induce frequency change indicating a saturated uniform layer formation with the first application (Figure 4). To investigate whether the MPBs of the half and the quarter MPB-modified KLFC LiDps are indeed toposelectively modified in a protein cage as illustrated in Figure 2, we introduced either the half (blue arrow) or quarter (red arrow) MPB-modified KLFC LiDps as a second layer and monitored frequency changes upon subsequent deposition of streptavidin, followed by deposition of the fully MPBmodified KLFC LiDps (Figure 4B,C). If biotin moieties are distributed only over a topologically limited region of the spherical protein cages, most of the biotins should bind to streptavidins which are anchored to the previous layer and become unavailable to mediate further layer formation (Figure 4, insets). Therefore, the introduction of either the half or quarter MPB KLFC LiDps as a second layer would be expected to alter or block the formation of subsequent layers of streptavidin and full MPB KLFC LiDps (Figure 4, insets). While frequency changes accompanying multilayer formation were almost identical for full, half, or quarter MPB KLFC LiDps up to the second layer, there were pronounced differences upon subsequent addition of streptavidin and full 2363

Figure 4. QCM resonance frequency change (-∆F) profiles of alternate MPB KLFC LiDps protein cages (down double arrow) and streptavidin (down arrow) on the gold QCM sensors. (A) The alternate depositions of full MPB KLFC LiDps protein cages (down double arrow) and streptavidin (down arrow). (B) The alternate depositions of full MPB KLFC LiDps protein cages (down double arrow) and streptavidin (down arrow) with the half MPB KLFC LiDps protein cages (blue, down double arrow) as a second layer. (C) The alternative depositions of full MPB KLFC LiDps protein cages (down double arrow) and streptavidins (down arrow) with the quarter MPB KLFC LiDps protein cages (red, down double arrow) as a second layer. (D) Plot of resonance frequency changes (-∆F) of full (black), half (blue), and quarter (red) MPB KLFC LiDps as a second layer at each layer deposition. (Insets) circle, protein cage; square, streptavidin.

MPB KLFC LiDps to the third layer (Figure 4). Streptavidin depositions on multilayers composed of half or quarter MPB KLFC LiDps (Figure 4B,C) resulted in frequency changes that were approximately 35 and 80% less, respectively, than streptavidin depositions on multilayers composed of full MPB KLFC LiDps (Figure 4D). There was a striking difference in the frequency reduction accompanying the final deposition of full MPB KLFC on multilayers formed from the half and quarter MPB KLFC LiDps (Figure 4B,C,D). While the deposition of full MPB KLFC LiDps on mutlilayers constructed from full or half MPB KLFC LiDPS were nearly identical (Figure 4B,D), the deposition on mutlilayers constructed from quarter MPBmodified KLFC LiDps was almost completely blocked. This result implies, as described in Figure 2, that MPBs are toposelectively attached near the 3-fold axis of the quartermodified KLFC LiDps and the spatially limited distribution of attached biotins only allows a localized single direction interaction to the underlying streptavidin layer upon deposition. The homogeneous orientation and close packing of quarter MPB KLFC LiDps likely results in this nearly complete blocking of subsequent full MPB KLFC LiDps 2364

deposition (Figure 4C, inset). In contrast, hemispheres of half MPB KLFC LiDps can be exposed to the next streptavidin layer as well as interact with the bottom streptavidin layer, because a protein cage is a roughly spherical threedimensional object and arrangements of deposited protein cages can be situated as illustrated in the cartoon (Figure 4B, inset). To ensure uniform LbL formation of Janus-like protein cages, we investigated the surface roughness of each layer formed on the QCM sensors using atomic force microscopy (AFM). Each layer was relatively flat, and localized cluster formation was not observed (Figure F5 in Supporting Information). However, the surface of the QCM sensor itself was quite rough (Figure F5 in Supporting Information) making interpretation ambiguous. Therefore, LbL assemblies of MPB-modified KLFC LiDps were further characterized by AFM on mica surfaces (Figure 5). The first layer of MPBmodified KLFC LiDps was formed by electrostatic interactions between the negative charged mica surface and MPBmodified KLFC LiDps, which are densely populated with surface exposed lysine residues. Deposition of MPB-modified KLFC LiDps resulted in a uniform monolayer exhibiting Nano Lett., Vol. 9, No. 6, 2009

Figure 5. AFM surface and depth profiles of protein cage LbL assemblies. Height images of (A) mono-, (B) bi-, and (C) trilayer KLFC LiDps assemblies on mica. Craters were scraped into each sample and assembly depths were determined from line scans (insets and lower panels, respectively). Average depths for each assembly are presented in (D). Error bars represent standard deviations (n ) 9 measurements for each sample).

features of protein cages (Figure 5A, top panel). Surface roughness data corroborated that deposition proceeded without clustering (Table T1 in Supporting Information). Successive layer formations were achieved by alternate depositions of streptavidin- and MPB-modified KLFC LiDps identical to the QCM experiment. Notably, surfaces were similarly uniform with no significant clustering (Figure 5B,C, top panel). The slight increases in surface roughness observed for both bi- and trilayer assemblies most likely resulted from spatial mismatch between streptavidins (4 nm, cubic) and protein cages (9 nm, spherical) (Table T1 in Supporting Information). AFM was also used to quantify the thickness of the assemblies and to confirm that protein cage deposition was uniform (Figure 5A-C, bottom panels). To this end, craters were formed by rastering the AFM probe across sample surfaces in contact mode and surfaces were reimaged in tapping mode (Figure 5, insets). Three independent craters were generated in each mono-, bi-, and trilayered assembly to evaluate the uniformity of layers, and the depths of each crater were averaged from three independent line scans on three different positions (Table T2 in Supporting Information). Small deviations of the crater depth in each assembly indicate that LbL formation was uniform, consistent with surface roughness measurements. Average depths of layers increased linearly as additional layers were deposited (Figure 5D) corroborating the QCM measurements. However, the measured layer thicknesses were lower than expected values based on the diameter of protein cages (9 nm). These differences may reflect the dehydrated form of protein cages differing from hydrated form or distortions in the cage architecture due to tight interfacial interactions between layers (e.g., between both mica/cage and streptavidin/MPBmodified cage). In this study, we have generated Janus-like protein cages using the approach of toposelective surface modification on solid supports and determined their localized distributions precisely at the molecular level using mass spectrometry and LbL formation. Spatially controlled surface presentation of multiple functional groups on a protein cage will enable them to be used as polarized nanoscale building blocks for directed hierarchal assembly, as well as for fabricating nanoscale Nano Lett., Vol. 9, No. 6, 2009

cargoes having distinct functional groups on each hemisphere. In addition to the exterior surface, protein cages provide an interior cavity that can be used for spatially constrained nanoparticle synthesis, encapsulation of preformed nanomaterials, or covalent attachment of organic molecules, such as drugs and therapeutic reagents.25,34-37,42-47 Thus, the addition of nanoscale Janus-like protein cages to the repertoire of nanoscale building blocks will expand the possibilities for fabricating nanostructured functional materials. Acknowledgment. We thank Dr. Brian Bothner and Vamseedhar Rayaprolu for assistance in QCM measurements. This research was supported in part by grants from Human Frontier Science Program (RGP61/2007), the National Science Foundation (CBET-0709358), Office of Naval Research (N00014-03-1-0692) and the Department of Energy (DE-FG02-07ER46477). Supporting Information Available: Details on experimental procedures, SEC data, mass spectra of F5 M and MPB treated KLFC LiDps, control QCM measurements, AFM images of LbL formation on the QCM sensors, and detailed AFM results of LbL formation on mica. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Cheung, C. L.; Camarero, J. A.; Woods, B. W.; Lin, T.; Johnson, J. E.; De Yoreo, J. J. J. Am. Chem. Soc. 2003, 125, 6848. (2) McMillan, R. A.; Howard, J.; Zaluzec, N. J.; Kagawa, H. K.; Mogul, R.; Li, Y.-F.; Paavola, C. D.; Trent, J. D. J. Am. Chem. Soc. 2005, 127, 2800. (3) Nam, K. T.; Wartena, R.; Yoo, P. J.; Liau, F. W.; Lee, Y. J.; Chiang, Y. M.; Hammond, P. T.; Belcher, A. M. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 17227. (4) Huang, Y.; Chiang, C.-Y.; Lee, S. K.; Gao, Y.; Hu, E. L.; Yoreo, J. D.; Belcher, A. M. Nano Lett. 2005, 5, 1429. (5) Nam, K. T.; Kim, D. W.; Yoo, P. J.; Chiang, C. Y.; Meethong, N.; Hammond, P. T.; Chiang, Y. M.; Belcher, A. M. Science 2006, 312, 885. (6) Miura, A.; Hikono, T.; Matsumura, T.; Yano, H.; Hatayama, T.; Uraoka, Y.; Fuyuki, T.; Yoshii, S.; Yamashita, I. Jpn. J. Appl.Phys. 2006, 45, L1. (7) Miura, A.; Tsukamoto, R.; Yoshii, S.; Yamashita, I.; Uraoka, Y.; Fuyuki, T. Nanotechnology 2008, 19, 255201. (8) Miura, A.; Uraoka, Y.; Fuyuki, T.; Yoshii, S.; Yamashita, I. J. Appl. Phys. 2008, 103, 74503. 2365

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Nano Lett., Vol. 9, No. 6, 2009