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Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Metal-Assisted Assembly of Protein Containers Loaded with Inorganic Nanoparticles Matthias Künzle,† Thomas Eckert,‡ and Tobias Beck*,†,§,∥ †
Institute of Inorganic Chemistry, RWTH Aachen University, 52074 Aachen, Germany Institute of Physical Chemistry, RWTH Aachen University, 52074 Aachen, Germany § I3TM, RWTH Aachen University, 52074 Aachen, Germany ∥ JARA SOFT and JARA FIT, RWTH Aachen University, 52074 Aachen, Germany
Inorg. Chem. Downloaded from pubs.acs.org by NORTH CAROLINA A&T STATE UNIV on 10/20/18. For personal use only.
‡
S Supporting Information *
ABSTRACT: Protein containers are suitable building blocks for bioinorganic materials. Here, we show that high concentrations of magnesium ions induce the formation of a unitary protein scaffold, whereas low magnesium concentration leads to a binary protein scaffold. The molecular interactions in the protein scaffold were characterized with X-ray crystallography to high resolution. We show that the unitary framework can be applied for the assembly of inorganic nanoparticles such as metal oxides into highly ordered bioinorganic structures. Our work emphasizes the structural tunability of protein-container-based materials, important for adjusting emerging properties of such materials.
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involve hydrogen bonds and van der Waals contacts.23 In contrast, in supramolecular chemistry, designed interactions of metal ions with matching ligands allow crafting of sophisticated three-dimensional structures known as metal−organic frameworks (MOF) with a wide scope of applications.24 Hence, the introduction of metal-assisted assembly into protein scaffolds is intriguing because it has been shown that the introduction of defined binding sites for metal ions enables metal-assisted assembly of isolated protein chains into larger supramolecular structures.25 Moreover, by the addition of short organic linkers, ordered protein frameworks were constructed with protein containers as nodes, which are reminiscent of classical MOFs.26,27 A recent study shows that bivalent ions such as magnesium can induce the assembly of viral protein containers; however, detailed molecular interactions for the magnesium ions with the protein residues were not elucidated.28
INTRODUCTION Nanostructured materials offer exciting applications in the fields of catalysis, photon manipulation, sensor technology, and medicine.1−4 However, it remains difficult to control materials’ structural features both on the molecular and nanoscopic scale at the same time. Materials based on protein scaffolds can overcome this challenge: Proteins are produced by nature with atomic precision on the molecular scale in a programmed synthesis. Due to their nanometer dimensions, protein scaffolds also access larger scales, thus combining the atomic and nanoscale with high precision. In particular, protein containers, which consist of protein monomers, are a viable building block for materials: the container cavity can be used to synthesize or encapsulate inorganic cargo such as nanoparticles.5−15 Along these lines, we have used redesigned ferritin protein containers16 as building blocks to construct a binary protein scaffold. This framework provided a unique way to arrange inorganic particles into highly ordered binary threedimensional materials.17 We recently showed that the proteincontainer-based materials are suitable for catalysis: the nanoparticles are accessible within the porous material.18 Other approaches to construct crystalline bioinorganic materials use monomeric protein to form a proteins scaffold that is subsequently used for deposition of inorganic materials.19−22 Understanding the molecular interactions of the protein components on the atomic level is crucial for the construction of well-defined protein-based materials and the manipulation of these protein architectures. In this way, the properties of the resulting bioinorganic materials can be tuned. In crystals of unmodified proteins, interactions in the crystal lattice usually © XXXX American Chemical Society
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EXPERIMENTAL SECTION
A brief description of the experimental procedures follows. For a detailed description, please refer to the Supporting Information. Protein Crystallography. Suitable crystallization conditions were identified using commercial sparse matrix screening. Crystals of Ftn(neg) were obtained by increasing the magnesium concentration to 0.4−1.2 M magnesium acetate. Data were collected at the Swiss Light Source, Villigen, Switzerland. Data collection statistics and refinement details are shown in Table S1. Synthesis of Metal Oxide Nanoparticles within the Protein Containers. Metal oxide nanoparticles were synthesized by adding Received: July 18, 2018
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DOI: 10.1021/acs.inorgchem.8b01995 Inorg. Chem. XXXX, XXX, XXX−XXX
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Figure 1. Control of structure type with Mg2+ concentration. Shown are the molecular interactions at the containers’ interfaces. Right panels: Hydrogen bonds are shown as dotted lines. Containers are related by a twofold symmetry axis in the crystal unit cell, and symmetry-equivalent residues are denoted with #. Water molecules and the magnesium ion are shown as red and gray spheres, respectively. For clarity reasons, hydrogen bonds to water molecules are omitted if not involved in connecting the container interfaces. Residue labels include the chain letter in parentheses with mutated residues highlighted in red. Inset shows a single crystal composed of Ftn(neg) with scale bar of 50 μm.
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metal precursor salts (NH4)2Fe(SO4)2, (NH4)2Co(SO4)2 or CeCl3 as a solution to the protein solution. Simultaneously, hydrogen peroxide as oxidant was added. Nanoparticle−protein composites were purified by centrifugation, chromatography (size-exclusion), and sucrosegradient centrifugation. Transmission Electron Microscopy. Samples were applied to copper grids covered with Formvar and carbon (Ted Pella, 01814-F) by incubation, additionally with uranyl acetate for stained samples. All analyses were carried out with a Zeiss Libra 200 FE transmission electron microscope with energy filters and operated at 200 kV. Images were analyzed using the ImageJ software.29 Crystalline Protein-Nanoparticle Materials. Crystals of Ftn(neg) loaded with metal oxide nanoparticles were obtained using the same assembly conditions as for the empty containers. Crystal Fixation. Crystals were fixated in situ in the hanging drop of the crystallization setup. The cover slide with the crystals in the crystallization drop was briefly removed to add 10 μL of 25% glutaraldehyde to the reservoir solution (final concentration 0.5%). Small Angle X-ray Scattering (SAXS). SAXS data were obtained from fixated crystals in mother liquor, transferred to a borosilicate glass capillary (Hilgenberg GmbH, Germany). SAXS data were recorded with a S-Max3000 system equipped with a Rigaku MicroMax-002+ X-ray microfocus generator (Cu Kα radiation, λ = 1.54 Å) in a q range from 0.004−0.22 Å−1 at room temperature. Data were analyzed with SAXSGUI (Rigaku Innovative Technologies, Inc. and JJ X-ray Systems ApS). One-dimensional SAXS data were obtained by azimuthally averaging the 2D scattering data. The magnitude of the scattering vector q is given by q = 4π sin θ/λ, where 2θ is the scattering angle.
RESULTS AND DISCUSSION We recently used designed molecular interactions between two oppositely charged protein containers as a general method to assemble protein containers and the encapsulated nanoparticles into highly ordered binary structures.17,18 Because we did not observe any metal ions involved in contacts between the two charged protein containers, we were curious if interactions of protein residues with metal ions could give an additional handle to control the structure of such systems. In the present study, we used redesigned protein containers Ftn(pos) and Ftn(neg), which we had employed to construct a binary protein scaffold.17 Particularly, we were interested if high concentrations of metal ions could influence the structure type of the protein scaffold. The assembly conditions to obtain the binary protein lattice contain low concentration of magnesium ions in the range of 100−300 mM. Importantly, as noted above, no magnesium ions were present in the crystal structure within this concentration range. Interestingly, increasing the magnesium concentration first leads to a zone where no crystals grow. However, magnesium concentrations above 400 mM resulted again in crystal formation with a similar morphology as for low magnesium concentrations (see inset Figure 1). The structure of these crystals was determined by protein crystallography up to high resolution (1.6 Å; see Table S1 in Supporting Information for details on crystallographic data and refinement details). The protein crystal structure shows that, B
DOI: 10.1021/acs.inorgchem.8b01995 Inorg. Chem. XXXX, XXX, XXX−XXX
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eq S1, Supporting Information). According to this formalism, the concentration of magnesium ions factors in with the power of six. Therefore, doubling the magnesium concentration, which takes place when moving in the crystallization conditions from the binary to the unitary structure (200− 400 mM), should result in a nearly 64-fold increase of complex concentration. Although this exact concentration dependence remains to be experimentally tested, the strong effect will favor a protein−magnesium complex, hence formation of unitary crystals. The second effect is that increasing the concentration of magnesium and its counterion (acetate) alters the ionic strength of the crystallization solution. With increased ionic strength, the Debye length decreases. Interactions between oppositely charged containers, for example salt bridges at the Ftn(neg) and Ftn(pos) interface in the binary structures, are screened because of the presence of counterions. Similarly, other ionic interactions, e.g. at the Ftn(pos)/Ftn(pos) interface (Figure 1, bottom right), are screened as well. With higher salt concentration, these nonmetal ionic interactions are successfully screened, while at the same time, the more specific magnesium-aspartate coordination is favored. Overall, the coordination interface’s plasticity is crucial for realization of the different types of interactions. Please note: in addition to the magnesium ions required for complex formation in the unitary structure, additional magnesium ions are likely to be present. These ions bind transiently in solution to residues of the protein container. Whereas some ions are observable in the crystal structure (Figure S1 and S2), most ions will not be visible in the crystal structure due to site disorder. Presumably due to the bridging via cations instead of direct interactions, the distance between the centers of Ftn(neg) containers in the Ftn(neg) structure is longer than in the binary structure composed of Ftn(neg) and Ftn(pos) (128.0 vs 126.6 Å). For the binary crystallization condition at lower magnesium concentration, the role of the magnesium cations, although they are not directly observable in the crystal structure, is to tune the ionic strength to an optimal range where binary assembly is favored. Importantly, substitution of magnesium formate with sodium formate or acetate (monovalent cation) showed crystallization, but crystals had inferior quality and a different morphology (Figure S3). In protein crystallization, it is rather common that a specific composition of the crystallization condition is required for the formation of high-quality crystals, while the components will not show up in the final structure. To further investigate the self-assembly process of the two charged protein containers with respect to salt concentration, we conducted crystallization experiments with varied concentration of magnesium acetate (Figure 2 and Supporting Information) at protein concentrations of 4 mg/mL Ftn(neg) and 4 mg/mL Ftn(pos). At concentrations below 100 mM magnesium acetate, no screening of electrostatic interactions takes place due to low salt concentration and amorphous precipitate forms. At low concentrations (100−300 mM magnesium acetate), binary crystals are formed (for structural details, see Künzle et al.17). An intermediate zone exists between 300 and 400 mM where no crystals form due to the complete screening of interactions between Ftn(neg) and Ftn(pos). At concentrations between 400 and 1200 mM, crystals composed of only Ftn(neg) are formed, the unitary phase. Above 1200 mM magnesium concentration, sufficient magnesium cations are present, and thus, magnesium complexes with only one protein ligand instead of two are
indeed, magnesium ions are now found at the interfaces between protein containers. Intriguingly, the structure type changes from a binary structure, with contacts between oppositely and like-charged containers, to a unitary structure composed solely of Ftn(neg) containers. Here, the containers adopt the cubic space group P23 with unit cell parameter a = 181.0 Å (structure deposited with PDB ID 5JKK). Inspection of the molecular structure shows that the Ftn(neg) containers in the Ftn(neg) structure are bridged by Mg2+ ions (Figure 1, top panel). Each Mg2+ ion is located on a twofold symmetry axis of the crystal lattice and is coordinated by six oxygen atoms in an octahedral fashion. Amino acids aspartic acid and glutamine (residues Asp84 and Gln86) are present as ligands to cross-link the container interfaces. The Mg2+ ion sitting between two containers is directly coordinated at a distance of 2.1 Å by a carboxylate oxygen (syn-η1) from Asp84 (chain C) and by a carboxylate oxygen (syn-η1) from its symmetry mate. This distance falls within the range observed typically for magnesium coordination in protein structures.30 In addition, the magnesium ion is coordinated by four water molecules (distances 2.1 and 2.2 Å). Two of these water molecules, related by twofold symmetry, are linked to the protein containers via hydrogen bonds to Gln86. From the adjacent protein chains, a second Asp84 and its symmetry mate further link these two water molecules to the protein. Therefore, this network cross-links the container interfaces. The residues involved in this contact interface are important for interactions in the binary structure as well. In the crystal structure composed of both containers, Ftn(neg) and Ftn(pos) (Figure 1, bottom), residues Asp84 and Gln86 form contacts between containers, albeit without any metal ions. As described previously,17 contacts between two Ftn(neg) containers involve hydrogen bonds, mostly mediated by water molecules (Figure 1, bottom). In addition, there is a hydrogen bond between symmetry-related residues Gln83 (chain J). Contacts between two Ftn(pos) containers are composed of hydrogen bonds, for example between the newly introduced Lys90 (chain D) and symmetry equivalents of Asp84 (chain C) and Gln86 (chain D). In native ferritin crystal structures, residues Gln86 and Asp84 along with their symmetry equivalents are also the residues that mediate crystal contacts between ferritin containers with the help of divalent metal ions such as Cd2+ or Zn2+.31,32 However, instead of only one metal coordination site, two coordination sites at the contact interface are observed in those structures, with the carboxylate oxygen atoms of the residues coordinating the divalent metal ions (η2 for Asp84).32 This coordination environment is similar to the binary Ftn(neg)/Ftn(pos) structure: in place of the metal ions, the cationic residue Lys90 interacts with the residues (Figure 1). In addition to the magnesium ions found at the Ftn(neg)/ Ftn(neg) interface in the unitary Ftn(neg) structure, there are two more binding sites for these ions, but these are not relevant for the formation of the unitary structure (see Figures S1 and S2). The transition from the binary structure at low magnesium concentration to the unitary structure may be puzzling at first but can be rationalized with two effects taking place at the same time. The first one is that the increase of the magnesium ion concentration significantly impacts the formation of the protein−magnesium complex. Each ferritin container has 12 coordination sites for magnesium, with each metal ion shared between two adjacent protein containers. The complex formation may be described with the law of mass action (see C
DOI: 10.1021/acs.inorgchem.8b01995 Inorg. Chem. XXXX, XXX, XXX−XXX
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calcium (see Supporting Information for experimental details). Crystallization trials with zinc salts resulted in amorphous precipitate. For crystallization trials with calcium salts, a more structured precipitate was obtained; however, no crystals form in these conditions. As discussed above, sodium ions are not suitable to form high-quality crystals of the binary phase. Crystallization of the unitary phase with sodium salts fails completely. Therefore, the direct substitution of magnesium with these cations is not possible. Sodium as a monovalent ion does not form the aspartate−metal complex required for crystallization of the unitary phase. Furthermore, divalent calcium and zinc are not able to form the complex required for crystallization because they differ in ionic radii (Mg vs Ca) or complexation behavior (Mg vs Zn). However, crystallization of the unitary phase with magnesium salts can be carried out at a range of different pH values. Results from the robotic screening indicate that between pH 4.6 and 8.5, crystals of the unitary phase are formed (Figure S5). Here, the aspartate residues remain deprotonated and can form the complex with magnesium required for crystallization. As an application for the protein scaffold, we used the containers to assemble inorganic nanoparticles into highly ordered structures. We recently showed that metal oxide nanoparticles inside the protein container scaffold are still accessible for catalysis.18 Here, we show that the novel unitary scaffold can be used to arrange the nanoparticles into a highly ordered lattice. To this end, prior to assembly, we synthesized metal oxide nanoparticles inside the protein container using cobalt, iron, and cerium salts to obtain three composites CoFtn(neg), FeFtn(neg), and CeFtn(neg) (Figure 3, top). See Supporting Information for details on experimental procedures and characterization (Figure S6). The container pores enable access to the container cavity for metal precursor and the oxidant hydrogen peroxide. Protein− nanoparticle composites were purified by size exclusion chromatography. Assembly of these composites was carried
Figure 2. Crystallization diagram for the protein container system depicted in Figure 1. Formation of precipitate, the binary phase, and the unitary phase is observed.
formed. Here, cross-linking, as observed in the crystal structure of the unitary phase, does presumably not take place, and therefore, the protein remains soluble, resulting in clear crystallization drops. Based on additional crystallization experiments where the protein concentration was varied as well, we derived a rough crystallization phase diagram (Figure S4). As expected for a typical phase diagram in protein crystallization, the binary phase shows a zone with no nucleation (above 300 mM salt) at all protein concentrations, followed by a nucleation and growth zone for crystals at about 200 mM salt. At 100 mM, due to increased interactions between the oppositely charged containers, showers of crystalline precipitate appear, predominantly at higher protein concentration. Precipitate forms at low salt concentrations (see also Figure 2). For the unitary phase, crystallization takes place only at higher protein concentration where sufficient protein for complex formation with magnesium (see above) is available. To study the role of the metal ion identity, we conducted crystallization experiments by substituting magnesium in the acetate and formate salts with the divalent ions zinc and
Figure 3. Synthesis of crystalline protein−nanoparticle materials with Ftn(neg). Top: Nanoparticle synthesis inside Ftn(neg) and assembly using conditions as for empty Ftn(neg) (see Figure 1). Bottom: Optical microscopy pictures of representative crystals from Ftn(neg) with cargo loading: (A) CoFtn(neg), (B) FeFtn(neg), and (C) CeFtn(neg). (D) Negative control: Crystal grown in a drop composed of solution of iron-loaded Ftn(pos) and empty Ftn(neg) at high magnesium concentration. The lack of yellow color indicates a pure Ftn(neg) crystal formed instead of a binary crystal. Scale bars are 50 μm. D
DOI: 10.1021/acs.inorgchem.8b01995 Inorg. Chem. XXXX, XXX, XXX−XXX
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Figure 4. SAXS data of crystals composed of Ftn(neg) loaded with cobalt oxide nanoparticles. (A) A representative optical microscopy image of crystals used for SAXS analysis (scale bar: 100 μm). (B) 2D (left) and radially averaged 1D SAXS data (right) for crystals composed of Ftn(neg) and cobalt oxide nanoparticles. Experimental data are shown in black, and predicted scattering pattern is shown in red. A unit cell of the material is shown.
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CONCLUSIONS In conclusion, the protein scaffold used for the assembly of inorganic nanoparticles can be tuned between a unitary structure and a binary structure by adjusting the concentration of magnesium ions in the crystallization condition. In the unitary structure, the negatively charged protein containers Ftn(neg) are bridged by the magnesium ions. Shielding of interactions between oppositely charged containers and higher concentrations of magnesium ions available for the formation of the magnesium−protein complex favor in sum the unitary structure. These results prove the structures of protein container-based materials can be tuned without the need to alter the individual building blocks. Moreover, it is clear that metal ions play a crucial role in controlling the structure of protein scaffolds. For creation of functional nanoparticle materials, it is important to have different handles to tune the properties of the materials: one is changing the structural parameters. Here, we show that an additional protein matrix can be produced and used for the assembly of nanoparticles. Different structures of the nanoparticle superlattices could give rise to different emergent properties, e.g. for magnetic materials based on magnetic nanoparticles. Combining our approach of assembling nanoparticles using protein containers with the design of artificial metal binding sites will enable access to highly sophisticated materials. Potentially, the concentration of metal ions could induce reversible switching between different structures, thus dynamically changing the material properties.
out using conditions established for the formation of the unitary structure shown in Figure 1. Crystals composed of Ftn(neg), loaded with the three different types of nanoparticles, are shown in Figure 3, bottom. The crystals show similar morphology and sharp edges as crystals without nanoparticles. Crystals can be grown up to 500 μm in size. As negative control, crystals were grown in a drop composed of solution of iron-loaded Ftn(pos) and empty Ftn(neg) but with high magnesium concentration (0.5 M magnesium acetate in 100 mM Tris-HCl buffer pH 8.5 as reservoir solution). Here, selective growth of only the unitary Ftn(neg) structure is observed, as evidenced by the lack of yellow color in the final crystals. To characterize the nanoparticle superlattice, we used SAXS. Crystals were first fixated with glutaraldehyde. 33 This dialdehyde cross-links the protein scaffold (imine formation with lysines) and provides further stability to the materials, making handling for further analysis straightforward. Figure 4A shows a representative sample of Ftn(neg) crystals containing cobalt oxide nanoparticles. Crystals were characterized with scanning electron microscopy and EDX (Figure S7). For SAXS analysis (Figure 4B), measurements were carried out in a rotating sample tube to ensure that ensemble diffraction from the sample was recorded rather than Bragg peaks from only a few crystals that are by serendipity oriented in diffraction condition. 1D radially averaged SAXS data show the diffractogram for the nanoparticle superlattice. It is important to note that space group of the nanoparticle superlattice can be derived from the chiral P23 space group of the protein scaffold by adding centrosymmetry due to the spherical symmetry of the nanoparticles. Hence, the space group of the nanoparticle superlattice is Pn3̅. A predicted scattering pattern (red line in Figure 4B, right), using the position of the peak corresponding to reflection (111) for the calculation of the unit cell dimensions, is in accordance with the experimental data (black line in Figure 4B, right). The unit cell dimensions are 184.1 Å, similar to 181.0 Å of the protein scaffold determined by protein crystallography (Figure 1 and Table S1). The shrinkage in the cell parameters obtained for the X-ray crystal structures compared to the cell parameters for SAXS data can be readily rationalized with the decreased temperature during single crystal data collection (100 K vs room temperature for SAXS).34 A similar effect was observed in the binary nanoparticle structures.17 In comparison with the tetragonal unit cell found for the binary structure, no peaks preceding the main peak (111) are found, indicative of the cubic space group.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b01995.
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Details of experimental procedures and additional tables and figures (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Tobias Beck: 0000-0001-7398-3982 Notes
The authors declare no competing financial interest. E
DOI: 10.1021/acs.inorgchem.8b01995 Inorg. Chem. XXXX, XXX, XXX−XXX
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ACKNOWLEDGMENTS The authors thank Birgit Hahn for SEM data collection, Prof. Ulrich Schwaneberg for generous support with regard to protein production, Prof. Walter Richtering for SAXS access, Prof. Fitter for access to a crystallization robot, and Prof. Ulrich Simon for general support and helpful discussions. This work was generously supported by a Liebig scholarship to T.B. (Fonds der Chemischen Industrie), a doctoral scholarship to M.K. (Fonds der Chemischen Industrie), the DFG, and the Excellence Initiative of the German federal and state governments (I3TM Seed Fund grant and I3TM Step2Project grant to T.B.).
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