Self-Assembly of Protein Crystals with Different Crystal Structures

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Self-Assembly of Protein Crystals with Different Crystal Structures Using Tobacco Mosaic Virus Coat Protein as a Building Block Jianting Zhang, Xiao Wang, Kun Zhou, Gang Chen, and Qiangbin Wang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b08316 • Publication Date (Web): 19 Jan 2018 Downloaded from http://pubs.acs.org on January 21, 2018

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Self-Assembly of Protein Crystals with Different Crystal Structures Using Tobacco Mosaic Virus Coat Protein as a Building Block Jianting Zhang†‡, Xiao Wang§‡, Kun Zhou†, Gang Chen*§ and Qiangbin Wang*†¶ †

CAS Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine and i-Lab,

Suzhou Institute of Nano-Tech and Nano-Bionics, Chinese Academy of Sciences, Suzhou 215123, China ¶

School of Nano Technology and Nano Bionics, University of Science and Technology of

China, Hefei 230026, China §

School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210,

China

ABSTRACT: In this work, a typical cylinder-shaped tobacco mosaic virus coat protein (TMVCP) is employed as an anisotropic building block to assemble into triclinic and hexagonal close-packed (HCP) protein crystals by introducing cysteine residues at the 1 and 3 sites and four histidine residues at the C-terminal, respectively. The engineered functional groups of cysteine and histidine in the TMVCP and the self-assembly conditions determine the thermodynamics and kinetics in the self-assembly process for forming different crystal structures. The results show that the TMVCPs are thermodynamically driven to form triclinic crystals due to the formation of disulfide bonds between neighboring TMVCPs. On the other 1

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hand, the self-assembly of HCP crystals is kinetically directed by the strong metal–histidine chelation. This work not only greatly expands TMVCP for fabricating promising nanomaterials but also represents an approach to adjusting the protein crystal structures by tuning the thermodynamics and kinetics during crystallization.

KEYWORDS: TMV coat protein, self-assembly, protein crystal, crystal structure, protein interactions

The crystallographic superlattice synthesized from nanoscale “atom equivalents” is of particular interest for the creation of promising materials. The emergent and collective properties from these superlattices have shown great potential in many applications, such as optics, electronics, catalysis, and biomaterials.1-8 Inorganic nanoparticles are the most prevalent building block and have been used to assemble functional superlattices by controlling the constituent material into single,9 binary, or ternary10 assemblies.11 Furthermore, with the help of DNA nanotechnology, pre-designed crystal structures and adjustable cell parameters were exhibited in DNA-mediated nanoparticle superlattices, suggesting the potential to more intelligently fabricate periodic superstructures.12-15 More recently, the “atom equivalent” concept has been expanded from inorganic nanoparticles to protein motifs to construct biomolecular crystals with diverse chemical and structural properties.16-18 To date, various strategies have been employed to successfully construct protein crystals with tunable crystal structures, including electrostatic interaction,19 metal coordination,20,21 the symmetry-matching fusion method,22 dual interactions of protein–sugar interaction, π−π stacking,23 etc. However, it is worth noting that most of the

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current strategies are limited to using spherical protein motifs as building blocks to control the spatial arrangement and orientation of the as-prepared protein crystals, such as spherical Cowpea Chlorotic Mottle virus (CCMV),24,25 ferritin26 and oblate catalases27. Fabricating protein crystals from nonspherical building blocks remains a challenge due to their chemical and structural anisotropy and thus the complexity involved in their arrangement and orientation into periodic structures, particularly crystals with diverse crystal structures using identical protein building blocks. In this paper, we use the cylinder-shaped tobacco mosaic virus coat protein (TMVCP) as an anisotropic building block to construct protein crystals with different crystal structures by controlling the thermodynamics and kinetics of the self-assembly process. For this purpose, as shown in Figure 1, two TMVCP variants are constructed by introducing two cysteine or four histidine residues at the lateral surface of TMVCP, which are denoted by T103C-TMV1,3cys and T103C-TMV4his, respectively (Figure S1). The thiol and imidazole ligands in the mutated TMVCPs will bridge discrete TMVCPs via disulfide bond formation and metal–histidine chelation, thus enabling us to control the dynamic assembly process based on synergistically regulating the assembly thermodynamics and kinetics, thereby resulting in highly ordered protein crystals with different crystal structures. Significantly, T103C-TMV1,3cys CPs were assembled at a low temperature (4 °C) to slow down the assembly dynamics. Thus, this sample required incubation for one month to form triclinic crystals (Figure 1C). To facilitate the self-assembly of T103C-TMV4his CPs, Zn2+ was added to rapidly drive the TMVCPs to form stable crystals by strong metal–histidine chelation, resulting in hexagonal close-packed (HCP) TMV crystals (Figure 1E). 3

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RESULTS AND DISCUSSION

Figure 1. Schematic illustration of the self-assembly of TMVCPs into crystals with different crystal structures driven by the formation of disulfides bonds or metal–histidine chelation. (A) Molecular model of a pristine TMVCP. (B) Model of a cysteine-mutated CP (T103C-TMV1,3cys), onto which cysteine residues were introduced at the 1 and 3 sites, as shown in blue. (C) Model of triclinic TMV crystal assembled at 4 °C via disulfide-bond-induced

crystallization.

(D)

Model

of

histidine-mutated

CP

(T103C-TMV4his), onto which four histidine residues were inserted at the C-terminal, as shown in red. (E) Model of a HCP TMV crystal assembled via metal–histidine chelation.

As shown in Figure 2A-E, simple genetic mutations do not change the overall structures of T103C-TMV1,3cys and T103C-TMV4his CPs, which were verified by transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements (Figure S2). The small-angle X-ray scattering (SAXS) fitting indicated that under our experimental conditions, the TMVCPs had a diameter of 17.6 nm and a height of 10.6 nm using a hollow cylinder model, which is consistent with previous reports (Figure 2F).28,29

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Figure 2. Characterization of the two mutated TMVCPs by TEM, DLS and SAXS measurements. Schematics of (A) T103C-TMV1,3cys and (B) T103C-TMV4his, respectively. TEM images of (C) T103C-TMV1,3cys and (D) T103C-TMV4his CPs. (E) DLS results and (F) SAXS characterization of the two mutated TMVCPs.

Then, the assembly of T103C-TMV1,3cys CPs was performed at 4 °C for about one month to enable the crystallization process. In contrast, the self-assembly of T103C-TMV4his CPs was carried out at room temperature by adding Zn2+ to crosslink the TMVCPs into ordered structures. TEM and cryo-electron microscopy (cryo-EM) were employed to characterize the morphologies and crystal patterns (Figure 3). Figure 3A-C show the triclinic crystals assembled from T103C-TMV1,3cys CPs, which are hundreds of nanometers in size and prone to assembling in a polygonal geometry with sharp boundaries (Figure S3), reflecting the thermodynamic-dependent equilibrium of the assembled triclinic crystals.30,31 Meanwhile, 5

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the strong chelation between Zn2+ and the imidazole group of histidine drives T103C-TMV4his CPs to assemble into highly ordered HCP crystals with sizes ranging from hundreds of nanometers to several micrometers (Figure 3D-E). We speculated that the rapid reaction kinetics dominates the assembly process, therefore generating compact HCP crystals with more irregular morphologies than that of the triclinic T103C-TMV1,3cys crystals (Figure S4).

Figure 3. TEM and cryo-EM characterizations of the assembled TMV crystals. (A) Low-magnification

TEM

disulfide-bond-induced

image

of

crystallization.

T103C-TMV1,3cys (B)

crystal

High-magnification

assembled

TEM

image

via of

T103C-TMV1,3cys crystal with the computed Fourier transforms of the high-magnification image (inset). (C) Cryo-EM image of T103C-TMV1,3cys crystals. (D) Low-magnification TEM image of T103C-TMV4his crystal mediated by Zn2+–histidine chelation. (E) High-magnification TEM image of T103C-TMV4his crystal with the computed Fourier transforms of the high-magnification image (inset). (F) Cryo-EM image of T103C-TMV4his crystals.

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As depicted in Figure 3, the periodic TMVCP crystals with different crystal structures are achieved by engineering different protein–protein interactions and controlling the self-assembly conditions. However, TMVCPs with no cysteine or histidine residues were not found to crystallize after following the same crystallization procedures (Figure S5). These observations confirm the proposed roles of the designed disulfide bond and metal–histidine chelation in the self-assembly of TMV crystals. We further employed SAXS to investigate the crystalline features of these protein crystals and evaluate their degree of ordering. As shown in Figure 4A, for the T103C-TMV1,3cys crystals, the X-ray diffraction peak positions in the azimuthally integrated scattering profile are found at the wavevector transfer q = 0.31, 0.38, 0.44, 0.50, 0.53, and 0.62 nm-1, which correspond to Bragg reflections from the triclinic —

crystal planes with Miller indices (hkl) = (100)/(010), (001), (011)/(101), (110), (101), and (2 — —



11)/(210)/(200), respectively, as determined by the theoretical model simulation. The angles between the primitive vectors are found to be α = 73.12°, β = 105.51°, and γ = 108.95°, as shown in Figure 4A. The lattice constants a and b are confirmed to be 21.62 nm and 21.74 nm, respectively, which agree well with the results of the Fourier transform analysis of the T103C-TMV1,3cys crystals (Figure 3B, inset). The lattice constant c is 17.96 nm. As for the metal–histidine-chelation-mediated T103C-TMV4his crystals, as shown in Figure 4B, the positions of the diffraction peaks in the azimuthally integrated curve are found at q = 0.34, 0.38, 0.42, 0.67, 0.76 and 0.78 nm-1, which are related to the crystal planes with Miller indices (hkl) = (002), (100), (101), (110), (200) and (201), respectively, corresponding to a HCP structure. The lattice constants a and b are both confirmed to be 19.02 nm, which also

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agree well with the results from the Fourier analysis of T103C-TMV4his crystals (Figure 3E, inset). The lattice constant c is 38.00 nm.

Figure 4. SAXS characterization of the assembled TMV crystals. The experimental and simulated SAXS curves of the crystals in solution formed by (A) T103C-TMV1,3cys and (B) T103C-TMV4his CPs. The right panels show schematic diagrams of the crystal structures and their unit cell parameters.

To elucidate the mechanisms by which TMVCPs self-assemble into ordered crystals, the self-assembly process was carefully monitored via TEM and SAXS (Figure 5). For T103C-TMV1,3cys CPs, a slow stacking and arrangement procedure was required for the individual TMVCPs to self-assemble into large triclinic crystals (Figure 5A-C, G). In detail, the T103C-TMV1,3cys CPs were dispersed in 400 mM phosphate buffer (PB) at pH 5.0.

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After incubating for one day at 4 °C, random TMVCP aggregates accompanied by TMV nanorod bundles formed (Figure S6). After removing the TMV nanorod bundles by centrifugation, the T103C-TMV1,3cys CP aggregates were further incubated at 4 °C for ten days to form TMV crystal nuclei (Figure 5A). These nuclei served as seeds for the epitaxial growth of TMVCPs into large arrays (Figure 5B), and finally, well-organized triclinic crystals were assembled after a one-month incubation at 4 °C (Figure 5C). This process was validated by SAXS data, as shown in Figure 5G, in which more and stronger diffraction peaks appeared with the increasing incubation time, indicating the gradually improving structural order of the T103C-TMV1,3cys assemblies. Interestingly, when the triclinic crystals were further incubated at room temperature, HCP T103C-TMV1,3cys crystals were obtained, indicating the important role of temperature in determining the thermodynamics of T103C-TMV1,3cys CP assembly and the unit cell structure of the assembled crystals (Figure S7). The effect of temperature on the assembled protein crystals was further investigated in one of our previous reports,32 in which TMV tube bundles were assembled from T103C-TMV1,3cys CPs at room temperature under the same buffer conditions of pH 5.0 and 400 mM PB. Lowering pH or increasing the ionic strength of the incubation solution is well known to improve the growth of TMVCPs into tubes because of the reduction in the electrostatic repulsion between TMVCPs. Therefore, the T103C-TMV1,3cys CPs assembled into bundle structures with the formation of disulfide bonds along the x–y plane and z axis direction at a low pH and in a high-ionic-strength PB solution. However, decreasing the temperature to 4 °C greatly inhibited the oxidization of the thiol groups at the 1, 3, and 103 sites of TMVCPs into interparticle disulfide bonds. Thus, the metastable triclinic protein 9

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crystals assembled during a very slow process requiring up to one month. This process included three steps: seeding, epitaxial growth, and re-arrangement (Figure 5).

Figure 5. Crystal evolution of T103C-TMV1,3cys and T103C-TMV4his CPs. (A-F) Representative TEM images of the assemblies of T103C-TMV1,3cys CPs after (A) 10 days, (B) 20 days and (C) 30 days at 4 °C, and assemblies of T103C-TMV4his CPs after (D) 1 hour, (E) 12 hours and (F) 24 hours. (G-H) SAXS patterns of assemblies after different incubation times: (G) T103C-TMV1,3cys and (H) T103C-TMV4his CPs.

In contrast with T103C-TMV1,3cys CPs, which required a long incubation time to assemble into crystals, T103C-TMV4his CPs were driven by metal–histidine chelation to 10

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rapidly and efficiently self-assemble into highly ordered HCP crystals. TEM images showed that individual T103C-TMV4his CPs instantly packed together immediately after Zn2+ was added (Figure 5D). The strong coordination between imidazole groups and the bivalent cations facilitated the assembly of T103C-TMV4his CPs along the x–y plane, resulting in HCP T103C-TMV4his crystals (Figures 5E and 5F), which was in accordance with the SAXS results wherein diffraction peaks and intensities of HCP patterns appeared and became stronger along with the incubation time (Figure 5H). In addition, other divalent cations like Cu2+ could also be used as a chelating agent to assemble T103C-TMV4his CPs into HCP crystals (Figure S8).

CONCLUSION In summary, we successfully fabricated two types of TMV crystals, triclinic and HCP, by designing the functionalities of the anisotropic cylinder-shaped TMVCPs and finely modulating the thermodynamics and kinetics of TMVCP assembly. Specifically, cysteine and histidine residues were site-selectively introduced onto the outside surface of TMVCPs, which facilitated the horizontal assembly of TMVCPs into lattices via disulfide bonds formation and metal–histidine chelation, respectively. The thermodynamics-dependent assembly of T103C-TMV1,3cys CPs induced by disulfide bond formation slowly formed triclinic crystals during a month-long incubation at 4 °C, while the fast, kinetically controlled assembly of T103C-TMV4his CPs driven by strong metal–histidine chelation gave rise to the formation of HCP lattices within a 24-hour incubation. We expect that our strategy of designing the interactions between protein motifs and controlling the assembly conditions to

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modulate their assembly thermodynamics and kinetics will inspire the fabrication of functional supramolecular materials.

EXPERIMENTAL METHODS 1. Preparation of the TMV coat protein variants. In this paper, both T103C-TMV1,3cys and T103C-TMV4his variants were mutated based on the T103C-TMVCP. T103C-TMVCP contains a cysteine mutation at 103 site of TMVCP subunit (T103C) to form S-S bond in the cavity, which has been verified to dramatically improve the assembly efficiency and structural stability of the TMV coat protein in our early work.33 The mutagenesis of T103C-TMV1,3cys and T103C-TMV4his were made by routine polymerase chain reaction (PCR). The primers of T103C-TMV1,3cys were (P1: 5'-GAGATATACATATGTGTTATT GCATTACC-3' and P2: 5'-GGAATTCTCAGGTCGCCGGGCCG-3') and the primers of T103C-TMV4his were (P1: 5'-GAGATATACATATGAGCTATAGCAT-3' and P2: 5'-GGA ATTCTCAATGATGATGATGGGTCGCCGGGCCG-3'). The genes of the mutants were then cloned into the pET32a(+) vector (Novagen) at NdeI-EcoRI restriction sites using T4 DNA ligase, respectively. The constructs were verified by sequencing (Shanghai Invitrogen Biotechnology Co., Ltd.) and then transformed into E. coli BL21 (DE3) strain (Novagen). T103C-TMV1,3cys and T103C-TMV4his subunits were expressed and purified according to established methods.32,33 2. Assembly and purification of T103C-TMV1,3cys and T103C-TMV4his coat proteins. T103C-TMV1,3cys subunit resolution was dialyzed against PB (100 mM, pH 7.0) and incubated for 1 day at room temperature. Then, sucrose density gradient centrifugation (SDGC) was employed to separate T103C-TMV1,3cys coat proteins from other components. 12

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Briefly, the samples were loaded onto a 10-50% sucrose density gradient, and then centrifuged at 38000 rpm at 4 oC for 3.5 h. Gradient was collected at the third fraction of SDGC tube which was averagely divided into 10 fractions from top to bottom, and dialyzed against PB (100 mM, pH 7.0). The sample was concentrated to 5 mg/mL by the 30 KD centrifugal filter unit. As to the T103C-TMV4his coat protein, T103C-TMV4his subunit resolution was dialyzed against MOPS (20 mM, pH 7.0) containing 100 mM NaCl and incubated for 1 day at room temperature and subjected to the same procedure of SDGC. The sample was dialyzed against MOPS (20 mM, pH 7.0) containing 100 mM NaCl and concentrated to 5 mg/mL by the 30 KD centrifugal filter unit. 3. Assembly of TMVCP triclinic-packed and HCP crystals. For the triclinic-packed lattices assembly, 5 mg/ml T103C-TMV1,3cys coat protein resolution was dialyzed against PB (400 mM, pH 5.0) for 1 day at 4 oC. Then, the precipitate was discarded and the supernatant was further concentrated though ultrafiltration and incubated at 4 oC for 1 month. For the HCP lattices assembly, 5 mg/mL T103C-TMV4his coat protein resolution was added a final concentration of 30 µM ZnCl2 or CuCl2 and gently shaken at room temperature for 1 day. 4. TEM characterization. A 10 µL portion of sample solution was applied to a carbon-coated copper grid, removed after 5 min with filter paper, and negatively stained for 3 min with 2% phosphotungstic acid. All samples were imaged on an FEI Tecnai 20 TEM operated at 200 kV. Images were recorded with a Gatan Ultra Scan 894 CCD camera and processed with the ImageJ software.

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5. SAXS characterization. The SAXS experiments were performed at beamline BL16B1 of Shanghai Synchrotron Radiation Facility (SSRF). The incident X-ray photon energy was 10 KeV (wavelength = 0.124 nm). The beam size was 0.4 mm x 0.5 mm. The distance between the sample and the detector was set to 1.96 and 5 m respectively, for accessing different regions of the reciprocal space. During the experiments, the TMV solutions (~100 µL) were sealed into a 2-mm thick Teflon cell with two thin Kapton windows. The two-dimensional SAXS patterns were collected using a Mar165 CCD detector with the pixel size of 80 µm x 80 µm. The one-dimensional SAXS curves were obtained by integrating along the equal wavevector transferring rings and normalized by the direct beam intensities and exposure times. The background scattering intensity was also subtracted. When the TMV coat proteins are well dispersed in solution, the X-ray scattering intensity can be expressed as

, where the

of the TMV coat protein and

is the form factor

represents the orientational average. The hollow cylinder

model is chosen for the TMV coat protein to fit the SAXS data. The structural parameters of the TMV coat protein including its height, inner and outer diameters can be obtained. The structure factor

is introduced to fit the SAXS data from the TMV lattices. The

intensity can be expressed as34,35 ,

where

is a constant,

shape of TMV coat proteins.

is the Debye-Waller factor,

is related to the

in the equation accounts for the structural scattering,

which can be expressed as 14

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,

where

is the number of the TMV coat proteins in the unit cell,

peak-shape function,

,

and

the unit cell. The peak position

is the

are the fractional coordinates of the coat proteins inside is determined by the Miller indices (hkl) and the lattice

parameters of the TMV lattice. The SAXS data analyses are completed based on the self-developed codes in Matlab.

ASSOCIATED CONTENT

Supporting Information. Additional data including DLS data, TEM characterization. This material is available in the online version of this article at http://XXX(automatically inserted by the publisher).

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; [email protected] Author Contributions ‡

These authors contribute equally to this work

ACKNOWLEDGMENT This work was financially supported by the National Key Research and Development Program of China (2016YFA0101503), National Natural Science Foundation of China (No.

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21673280, 21425103, 11375256, U1632265). The authors thank all the team members of the beamline BL16B1 at Shanghai Synchrotron Radiation Facility (SSRF).

REFERENCES (1) Henry, E.; Dif, A.; Schmutz, M.; Legoff, L.; Amblard, F.; Marchi-Artzner, V.; Artzner, F. Crystallization of Fluorescent Quantum Dots within a Three-Dimensional Bio-Organic Template of Actin Filaments and Lipid Membranes. Nano Lett. 2011, 11, 5443-5448. (2) Zhou, Y. L.; Marson, R. L.; van Anders, G.; Zhu, J.; Ma, G. X.; Ercius, P.; Sun, K.; Yeom, B.; Glotzer, S. C.; Kotov, N. A. Biomimetic Hierarchical Assembly of Helical Supraparticles from Chiral Nanoparticles. ACS Nano 2016, 10, 3248-3256. (3) Yang, M.; Sun, K.; Kotov, N. A. Formation and Assembly-Disassembly Processes of ZnO Hexagonal Pyramids Driven by Dipolar and Excluded Volume Interactions. J. Am. Chem. Soc. 2010, 132, 1860-1872. (4) Singh, G.; Chan, H.; Baskin, A.; Gelman, E.; Repnin, N.; Kral, P.; Klajn, R. Self-Assembly of Magnetite Nanocubes into Helical Superstructures. Science 2014, 345, 1149-1153. (5) Kuzyk, A.; Schreiber, R.; Fan, Z. Y.; Pardatscher, G.; Roller, E. M.; Hogele, A.; Simmel, F. C.; Govorov, A. O.; Liedl, T. DNA-Based Self-Assembly of Chiral Plasmonic Nanostructures with Tailored Optical Response. Nature 2012, 483, 311-314. (6) Zhang, Z. L.; Tang, Z. Y.; Kotov, N. A.; Glotzer, S. C. Simulations and Analysis of Self-Assembly of CdTe Nanoparticles into Wires and Sheets. Nano Lett. 2007, 7, 1670-1675. 16

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(7) Saboe, P. O.; Lubner, C. E.; McCool, N. S.; Vargas-Barbosa, N. M.; Yan, H. J.; Chan, S.; Ferlez, B.; Bazan, G. C.; Golbeck, J. H.; Kumar, M. Two-Dimensional Protein Crystals for Solar Energy Conversion. Adv. Mater. 2014, 26, 7064-7069. (8) Li, L.; Wang, Q. Spontaneous Self-Assembly of Silver Nanoparticles into Lamellar Structured Silver Nanoleaves. ACS Nano 2013, 7, 3053-3060. (9) Murray, C. B.; Kagan, C. R.; Bawendi, M. G. Self-Organization of CdSe Nanocrystallites into 3-Dimensional Quantum-Dot Superlattices. Science 1995, 270, 1335-1338. (10) Shevchenko, E. V.; Talapin, D. V.; Kotov, N. A.; O'Brien, S.; Murray, C. B. Structural Diversity in Binary Nanoparticle Superlattices. Nature 2006, 439, 55-59. (11) Dong, A. G.; Ye, X. C.; Chen, J.; Murray, C. B. Two-Dimensional Binary and Ternary Nanocrystal Superlattices: The Case of Monolayers and Bilayers. Nano Lett. 2011, 11, 1804-1809. (12) Yu, Q. Y.; Zhang, X. N.; Hu, Y.; Zhang, Z. H.; Wang, R. Dynamic Properties of DNA-Programmable Nanoparticle Crystallization. ACS Nano 2016, 10, 7485-7492. (13) Park, S. Y.; Lytton-Jean, A. K. R.; Lee, B.; Weigand, S.; Schatz, G. C.; Mirkin, C. A. DNA-Programmable Nanoparticle Crystallization. Nature 2008, 451, 553-556. (14) Nykypanchuk, D.; Maye, M. M.; van der Lelie, D.; Gang, O. DNA-Guided Crystallization of Colloidal Nanoparticles. Nature 2008, 451, 549-552. (15) Jones, M. R.; Seeman, N. C.; Mirkin, C. A. Programmable Materials and the Nature of the DNA Bond. Science 2015, 347, 1260901.

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(16) Suzuki, Y.; Cardone, G.; Restrepo, D.; Zavattieri, P. D.; Baker, T. S.; Tezcan, F. A. Self-Assembly of Coherently Dynamic, Auxetic, Two-Dimensional Protein Crystals. Nature 2016, 533, 369-373. (17) Medalsy, I.; Dgany, O.; Sowwan, M.; Cohen, H.; Yukashevska, A.; Wolf, S. G.; Wolf, A.; Koster, A.; Almog, O.; Marton, I.; Pouny, Y.; et al. SP1 Protein-Based Nanostructures and Arrays. Nano Lett. 2008, 8, 473-477. (18) Luo, Q.; Hou, C. X.; Bai, Y. S.; Wang, R. B.; Liu, J. Q. Protein Assembly: Versatile Approaches to Construct Highly Ordered Nanostructures. Chem. Rev. 2016, 116, 13571-13632. (19) Kunzle, M.; Eckert, T.; Beck, T. Binary Protein Crystals for the Assembly of Inorganic Nanoparticle Superlattices. J. Am. Chem. Soc. 2016, 138, 12731-12734. (20) Brodin, J. D.; Carr, J. R.; Sontz, P. A.; Tezcan, F. A. Exceptionally Stable, Redox-Active Supramolecular Protein Assemblies with Emergent Properties. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 2897-2902. (21) Brodin, J. D.; Ambroggio, X. I.; Tang, C. Y.; Parent, K. N.; Baker, T. S.; Tezcan, F. A. Metal-Directed, Chemically Tunable Assembly of One-, Two- and Three-Dimensional Crystalline Protein Arrays. Nat. Chem. 2012, 4, 375-382. (22) Sinclair, J. C.; Davies, K. M.; Venien-Bryan, C.; Noble, M. E. M. Nat. Nanotechnol. Generation of Protein Lattices by Fusing Proteins with Matching Rotational Symmetry. 2011, 6, 558-562.

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(23) Sakai, F.; Yang, G.; Weiss, M. S.; Liu, Y. J.; Chen, G. S.; Jiang, M. Protein Crystalline Frameworks with Controllable Interpenetration Directed by Dual Supramolecular Interactions. Nat. Commun. 2014, 5, 4634. (24) Liljestrom, V.; Mikkila, J.; Kostiainen, M. A. Self-Assembly and Modular Functionalization of Three-Dimensional Crystals from Oppositely Charged Proteins. Nat. Commun. 2014, 5, 4445. (25) Kostiainen, M. A.; Hiekkataipale, P.; Laiho, A.; Lemieux, V.; Seitsonen, J.; Ruokolainen, J.; Ceci, P. Electrostatic Assembly of Binary Nanoparticle Superlattices Using Protein Cages. Nat Nanotechnol. 2013, 8, 52-56. (26) Liljestrom, V.; Seitsonen, J.; Kostiainen, M. A. Electrostatic Self-Assembly of Soft Matter Nanoparticle Cocrystals with Tunable Lattice Parameters. ACS Nano 2015, 9, 11278-11285. (27) Brodin, J. D.; Auyeung, E.; Mirkin, C. A. DNA-Mediated Engineering of Multicomponent Enzyme Crystals. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 4564-4569. (28) Forster, S.; Timmann, A.; Konrad, M.; Schellbach, C.; Meyer, A.; Funari, S. S.; Mulvaney, P.; Knott, R. Scattering Curves of Ordered Mesoscopic Materials. J. Phys. Chem. B. 2005, 109, 1347-1360. (29) Bhyravbhatla, B.; Watowich, S. J.; Caspar,. D. L. D. Refined Atomic Model of the Four-Layer Aggregate of the Tobacco Mosaic Virus Coat Protein at 2.4-Angstrom Resolution. Biophys. J. 1997, 74, 604-615.

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(30) Rad, B.; Haxton, T. K.; Shon, A.; Shin, S. H.; Whitelam, S.; Ajo-Franklin, C. M. Ion-Specific Control of the Self-Assembly Dynamics of a Nanostructured Protein Lattice. ACS Nano 2015, 9, 180-190. (31) Kitagishi, H.; Oohora, K.; Hayashi, T. Thermodynamically Controlled Supramolecular Polymerization of Cytochrome b(562). Biopolymers 2009, 91, 194-200. (32) Zhang, J. T.; Zhou, K.; Wang, Q. B. Tailoring the Self-Assembly Behaviors of Recombinant Tobacco Mosaic Virus by Rationally Introducing Covalent Bonding at the Protein-Protein Interface. Small 2016, 12, 4955-4959. (33) Zhou, K.; Li, F.; Dai, G. L.; Meng, C.; Wang, Q. B. Disulfide Bond: Dramatically Enhanced Assembly Capability and Structural Stability of Tobacco Mosaic Virus Nanorods. Biomacromolecules 2013, 14, 2593-2600. (34) Yager, K. G.; Zhang, Y.; Lu, F.; Gang, O. Periodic Lattices of Arbitrary Nano-objects: Modeling and Applications for Self-Assembled Systems. J. Appl. Cryst. 2014, 47, 118-129. (35) Senesi, A. J.; Lee, B. Small-Angle Scattering of Particle Assemblies. J. Appl. Cryst. 2015, 48, 1172-1182.

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