Letter pubs.acs.org/NanoLett
Designing Two-Dimensional Protein Arrays through Fusion of Multimers and Interface Mutations James F. Matthaei,† Frank DiMaio,‡ Jeffrey J. Richards,† Lilo D. Pozzo,† David Baker,‡,§ and François Baneyx*,† †
Department of Chemical Engineering, University of Washington, Seattle, Washington 918195, United States Department of Biochemistry, University of Washington, Seattle, Washington 918195, United States § Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, Maryland 20815, United States ‡
S Supporting Information *
ABSTRACT: We have combined fusion of oligomers with cyclic symmetry and alanine substitutions to eliminate clashes and produce proteins that self-assemble into 2-D arrays upon addition of calcium ions. Using TEM, AFM, small-angle X-ray scattering, and fluorescence microscopy, we show that the designed lattices which are 5 nm high with p3 space group symmetry and 7.25 nm periodicity self-assemble into structures that can exceed 100 μm in characteristic length. The versatile strategy, experimental approach, and hexagonal arrays described herein should prove valuable for the engineering of functional nanostructured materials in 2-D. KEYWORDS: Protein design, self-assembly, 2-D materials, bionanotechnology, artificial S-layers
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uilding blocks that self-assemble into periodic twodimensional (2-D) arrays with nanoscale precision are of considerable interest for the production of mesostructures with superior catalytic, transport, optoelectronic, or biological properties.1−5 Although the S-layers of archaea and bacteria are archetypal 2-D protein materials,6 and despite recent advances in the computational design of protein-based nanostructures,7−12 proteins remain underused for the fabrication of synthetic 2-D lattices.13,14 To engineer a protein monomer capable of robust self-assembly into regular 2-D arrays, we combined fusion of two copies of point-symmetric building blocks and sidechains truncations to eliminate clashes at the newly introduced interface (Figure 1). This strategy is a departure from the streptavidin−leucine zipper approach of Sinclair et al.,14 the metal-coordination approach of Tezcan and co-workers,13 and the helical-bundle-mediated approach of Lanci et al.12 We set out to identify a naturally occurring protein that could be modified to produce a flat, 2-D crystalline layer pierced by a single small pore. To this end, we evaluated the structure of oligomeric proteins15,16 deposited in the protein data bank (PDB) for the following design specifications: (i) C3, C4, or C6 rotational symmetry for easy tiling of a 2-D pattern; (ii) topologically smooth top and bottom faces to facilitate interactions with technological surfaces and interfaces including 2-D substrates; (iii) nonoccluded central pore smaller than 5 nm to allow for future templating of inorganic mineralization;17 (iv) flexible loops suitable for future insertion of solid binding peptides;18 and (v) termini oriented so that the C-terminus of one subunit in an oligomer can be fused to the N-terminus of a © XXXX American Chemical Society
Figure 1. Lead candidate identification. (a) Screen the PDB for oligomeric proteins with C3, C4, or C6 symmetry, flat faces and pore size 100 μm in characteristic length) thin structures with sharp edges and folded-over domains that we take to be self-assembled TTM sheets (Figure 4b and Supplementary Figure 7). Although free-floating selfassembled structures with surface areas greater than 1000 μm2 only represented ∼4% of the total (Supplementary Figure 8), long-range ordering at these dimensions is unparalleled for any self-assembling protein, including S-layer proteins. Short-range order was assessed by transmission electron microscopy (TEM) following negative staining of Ca2+supplemented TTM samples with uranyl acetate. TEM images revealed periodic hexagonal patterns and fringes associated with the stacking of multiple sheets (Figure 4c). Consistent with design predictions, we found that the unit cell was 7.25 nm by FFT analysis of micrographs (inset). AFM imaging on silicon substrates further revealed that self-assembled TTM was nearly flat with kinked edges and a height of 4.9 nm (Figure 4d−e). The smaller topographic features present on self-assembled terraces had the same height (Figure 4e) and may correspond to the stacking of independently nucleated monolayers. They may also be explained by a scenario in which an unpaired TM
Figure 5. SAXS analysis of wild type STM415 and self-assembled TTM. Smeared scattering intensity versus wave vector plots for a variant of S. typhimurium STM415 fitted with an N-terminal His-tag (a) and self-assembled TTM (b). Blue circles correspond to data and solid red lines to calculations. See text for details.
the wild type protein, a fit of the Guinier region of the SAXS profile yielded a radius of gyration of 34 ± 0.3 Å, a value in good agreement with the 30.9 Å calculated from the crystal structure when contributions from hexahistidine extensions are taken into account. Additionally, the scattering profile calculated from the 2GJV coordinates using CRYSOL25 matched experimental data with deviation at high Q attributable to the presence of His tags (Figure 5a, solid line). As seen from the lack of a Guinier region at low Q (Figure 5b), instrument resolution was insufficient to obtain the overall size of self-assembled TTM structures. However, high Q C
DOI: 10.1021/acs.nanolett.5b01499 Nano Lett. XXXX, XXX, XXX−XXX
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scattering intensities of STM415 and Ca2+-treated TTM were comparable, confirming that the hexameric structure of the wild type protein is preserved in the assembly. We did not observe sharp diffraction peaks at high Q that have been reported for certain S-layer proteins.26,27 This is likely due to a combination of the low resolution of our line-collimation instrument, poor signal-to-noise ratio, and thermal fluctuations that broaden and reduce the intensity of Bragg peaks. On the other hand, deconvolution of the STM415 form factor from that of the assembly yielded a power-law dependence of −2.1 ± 0.1 which is characteristic of a 2-D object (Figure 5 and Supplementary Figure 11). Finally, the scattering profile derived from the coordinates of the structure shown in Figure 2c was in very good agreement with experimental data with deviation at low Q indicating that the products of TTM self-assembly have a characteristic length exceeding that of the model (Figure 5b). Taken together, the above experiments validate design predictions: TTM self-assembles into flat 2-D arrays of with p3 symmetry (the asymmetry introduced by the linker makes the overall design p3 rather than p6 symmetric), 7.25 nm lattice constant, and 5 nm thickness that can grow to yield structures on the order of 100 μm. We expect that the TTM platform will be superior to S-layers for practical applications28,29 because of the lack of cell wall attachment domains and/or anisotropic faces; the flatness of its “top” and “bottom” surfaces; the availability of multiple solvent-exposed loops for insertion of solid binding peptides or other structural or functional elements; the presence of a single type of pore with welldefined and controllable dimensions to template inorganic synthesis; an ability to produce very large arrays and to redesign protomers to modulate assembly and/or surface chemistry; and more broadly, full genetic access for predictive engineering and re-engineering. Our design strategy should prove a valuable addition to the growing number of approaches being developed for the production of protein-based nanomaterials by enabling the construction of 2-D structures with exquisite control of space group symmetry, periodicity, and chemistry.
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ACKNOWLEDGMENTS We are grateful to Dr. Yongdong Liu for help with initial purification and reassembly experiments. Part of this work was conducted at the UW Molecular Analysis Facility, a member of the NSF-NNIN network and at EMSL, a DOE Office of Science User Facility at PNNL. This work was supported by NSF award BBBE 1401835 (to F.B.) and DOE BES DESC0005153 (to L.D.P.; SAXS experiments). J.F.M. acknowledges financial support from NIH through a Cancer Nanotechnology Training Grant (T32CA138312). D.B. and F.M. acknowledge financial support from DTRA and AFOSR.
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ASSOCIATED CONTENT
* Supporting Information S
Figure S1: Molecular surface of S. typhimurium STM4215. Figure S2: Engineering 2D self-assembly. Figure S3: TTM purification. Figure S4: Kinetics of TTM polymerization. Figure S5: Influence of the temperature on TTM assembly. Figure S6: Influence of cation identity on TTM assembly. Figure S7: Collision of two free-floating self-assembled TTM structures. Figure S8: Size distribution of self-assembled TTM structures. Figure S9: TEM images of Ca2+-treated TTM4. Figure S10: Fluorescent microscopy images of self-assembled TTM4 structures. Figure S11: Deconvolution of the form factors of self-assembled TTM. Complete experimental methods. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.5b01499.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest. D
DOI: 10.1021/acs.nanolett.5b01499 Nano Lett. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.nanolett.5b01499 Nano Lett. XXXX, XXX, XXX−XXX
