Self-Assembling Supramolecular Nanostructures Constructed from de

Research Article Cite This: ACS Synth. Biol. 2018, 7, 1381−1394

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Self-Assembling Supramolecular Nanostructures Constructed from de Novo Extender Protein Nanobuilding Blocks Naoya Kobayashi,†,§,⊥ Kouichi Inano,‡ Kenji Sasahara,† Takaaki Sato,‡,¶ Keisuke Miyazawa,# Takeshi Fukuma,# Michael H Hecht,∇ Chihong Song,○ Kazuyoshi Murata,○ and Ryoichi Arai*,†,§,∥,●,◇ †

Department of Applied Biology and ‡Department of Chemistry and Materials, Faculty of Textile Science and Technology, Department of Bioscience and Textile Technology, Interdisciplinary Graduate School of Science and Technology, Shinshu University, Ueda, Nagano 386-8567, Japan ⊥ Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan ¶ Center for Energy and Environmental Science, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Nagano, Nagano 380-8553, Japan # Division of Electrical Engineering and Computer Science, Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan ∇ Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States ○ National Institute for Physiological Sciences, Okazaki, Aichi 444-8585, Japan ∥ Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies, Tsurumi, Yokohama 230-0045, Japan ● Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, Matsumoto, Nagano 390-8621, Japan ◇ Department of Supramolecular Complexes, Research Center for Fungal and Microbial Dynamism, Shinshu University, Minamiminowa, Nagano 399-4598, Japan

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§

S Supporting Information *

ABSTRACT: The design of novel proteins that self-assemble into supramolecular complexes is important for development in nanobiotechnology and synthetic biology. Recently, we designed and created a protein nanobuilding block (PN− Block), WA20-foldon, by fusing an intermolecularly folded dimeric de novo WA20 protein and a trimeric foldon domain of T4 phage fibritin (Kobayashi et al., J. Am. Chem. Soc. 2015, 137, 11285). WA20-foldon formed several types of selfassembling nanoarchitectures in multiples of 6-mers, including a barrel-like hexamer and a tetrahedron-like dodecamer. In this study, to construct chain-like polymeric nanostructures, we designed de novo extender protein nanobuilding blocks (ePN− Blocks) by tandemly fusing two de novo binary-patterned WA20 proteins with various linkers. The ePN−Blocks with long helical linkers or flexible linkers were expressed in soluble fractions of Escherichia coli, and the purified ePN−Blocks were analyzed by native PAGE, size exclusion chromatography−multiangle light scattering (SEC−MALS), small-angle X-ray scattering (SAXS), and transmission electron microscopy. These results suggest formation of various structural homo-oligomers. Subsequently, we reconstructed hetero-oligomeric complexes from extender and stopper PN−Blocks by denaturation and refolding. The present SEC−MALS and SAXS analyses show that extender and stopper PN−Block (esPN−Block) heterocomplexes formed different types of extended chain-like conformations depending on their linker types. Moreover, atomic force microscopy imaging in liquid suggests that the esPN−Block heterocomplexes with metal ions further self-assembled into supramolecular nanostructures on mica surfaces. Taken together, the present data demonstrate that the design and construction of self-assembling PN−Blocks using de novo proteins is a useful strategy for building polymeric nanoarchitectures of supramolecular protein complexes. KEYWORDS: de novo protein, nanostructure, protein engineering, protein nanobuilding block, protein-based supramolecular polymers, self-assembly

A

ll organisms contain self-assembling biomolecules including proteins, nucleic acids, sugars, and lipids. The ability to design and control such assemblies is a central goal of biomolecular engineering, nanobiotechnology, and synthetic biology. The design and construction of artificial biomacromo© 2018 American Chemical Society

Received: January 5, 2018 Published: April 24, 2018 1381

DOI: 10.1021/acssynbio.8b00007 ACS Synth. Biol. 2018, 7, 1381−1394

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ACS Synthetic Biology

artificial and fusion proteins as nanoscale building blocks.1,24 These include nanostructures constructed from fusion proteins that were designed for symmetric self-assembly,25−31 computationally designed self-assembling protein nanocages with atomic level accuracy,6,32−35 and computationally designed β-propeller proteins.36,37 In addition, the design and controlled self-assembly of proteins into polymeric architectures via supramolecular interactions offers advantages in understanding the spontaneously self-assembling process and fabrication of various bioactive materials.38,39 These protein-based supramolecular polymers include chemically controlled polymeric self-assembly of protein nanorings,40 supramolecular hemeprotein polymers through heme−heme pocket interaction,41,42 3D domainswapped oligomers,43−45 self-assembling nanostructures constructed from designed coiled-coil peptide modules,46−50 the design of protein−protein interactions through modification of residues on intermolecular interfaces,51,52 and metal-directed self-assembling protein complexes.53−56 In the field of supramolecular chemistry, the chain length of supramolecular polymers can be controlled by the addition of a monofunctional monomer.57 This chain stopper or end-capper limits the polymerization or the growth of self-assembling bifunctional monomers. These examples of protein-based supramolecular polymers were also reported: supramolecular protein polymers via helix−helix association;58 supramolecular nanotube of chaperonin GroEL with length control using single-ring mutant as end-capper;59 and a series of supramolecular green fluorescent protein oligomers that were assembled in polygonal geometries (GFP nanopolygons).60 Recently, we designed and constructed the polyhedral protein nanobuilding block (PN−Block) WA20-foldon61 by fusing the intermolecularly folded dimeric de novo protein WA2023 with a trimeric foldon domain of T4 phage fibritin.62 The WA20-foldon formed several distinctive types of selfassembling nanoarchitectures from combinations of dimers and trimers in multiples of 6-mers (6-, 12-, 18-, and 24-mer), including a barrel-like-shaped hexamer and a tetrahedron-likeshaped dodecamer. The basic objective of the “PN−Block strategy” is to create various self-assembling nanostructures from a few types of simple and fundamental PN−Blocks. PN− Blocks comprising intermolecularly folded dimeric de novo proteins (e.g., WA20) as a key component for nanoarchitectures have the following advantages: (1) The simple, stable, and intertwined rod-like structure of the de novo PN− Block protein makes it easy to use for design and construction of simple and stable frameworks for nanoarchitectures,61 and (2) PN−Blocks that are based on simple binary patterning of de novo proteins have great potential for redesigning protein domains that function in vitro14−16 and in vivo.17−22 (“PN” in PN−Block has a different meaning from the polar and nonpolar abbreviations used in the binary code strategy for protein design.) Further investigations of new types of PN−Blocks and the reassembly of various PN−Blocks are essential steps for the development of the PN−Block strategy. Herein, we designed and created de novo extender protein nanobuilding blocks (ePN−Blocks) by tandemly fusing two de novo WA20 proteins with various linkers,63,64 and produced a new series of PN−Blocks that can be used to construct selfassembling extended or cyclized chain-like polymeric nanostructures (Figure 1). Moreover, to expand possibilities of the PN−Block strategy, we reconstructed heterooligomeric complexes by denaturating

lecules that self-assemble into supramolecular complexes are important steps toward achieving this goal. Proteins are the most versatile self-assembling biomacromolecules, which perform complex and functional tasks in all organisms. Protein functions are essentially determined by their three-dimensional (3D) structures, which are characterized into four hierarchical levels. Complex and refined structures create versatile functionalities of proteins. The design of a novel protein and complex is in essence an exploration of untracked areas of amino-acid sequence space. This exploration can be challenging, both because sequence space is vast and because the contribution of many cooperative and long-range interactions causes a significant gap between the primary structures and their resulting tertiary and quaternary structures. Research into de novo protein designs has progressed toward the construction of novel proteins,1,2 and has been achieved from (1) rational and computational designs,3−6 (2) combinatorial methods,7,8 and (3) semirational approaches that include elements of both.8−10 As a semirational approach, the binary code strategy was developed to design patterned polypeptide libraries (primary structures) for constructing tertiary structures of de novo proteins. Using secondary structure motifs with binary patterns of polar and nonpolar residues,9,10 de novo proteins with α-helixes and/or β-sheets have been successfully created.9−12 From a third-generation library of de novo 4-helix bundle proteins with binary patterns,13,14 several de novo proteins with functions in vitro14−16 and in vivo17−22 have been produced. We solved the crystal structure of the de novo protein WA20,23 which is a stable and functional de novo protein from the third-generation library.14 WA20 has an unusual dimeric structure with an intermolecularly folded (3D domainswapped) 4-helix bundle.23 Each WA20 monomer (“nunchaku”-like structure) comprises two long α-helices that are intertwined with the helices of another monomer (Figure 1A).

Figure 1. Schematics of extender protein nanobuilding blocks (ePN− Blocks). (A) Construction and homooligomeric self-assembly of ePN−Blocks. Ribbon representation and schematics of the intermolecularly folded dimeric de novo protein WA20 (PDB code 3VJF)23 are shown in red and blue. The ePN−Blocks were constructed by tandemly fusing two de novo WA20 proteins using various linkers.63,64 Helical and flexible linkers are shown as yellow rods and black lines, respectively. (B) Schematics of ePN−Blocks with helical linkers (HL). (C) Schematics of ePN−Blocks with flexible linkers (FL).

The WA20 structure is stable (melting temperature, Tm, about 70 °C) and forms a simple rod-like shape.23 Hence, the stable, simple, and unusual intermolecularly folded structure of the de novo protein WA20 can be applicable to basic framework tools in nanotechnology and synthetic biology. In these years, several approaches have been developed to design and construct self-assembling protein complexes using 1382


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ACS Synthetic Biology and refolding extender and stopper PN−Blocks. In addition, we demonstrate that the complexes can further self-assemble into supramolecular nanostructures with metal ions.

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RESULTS AND DISCUSSION Design of de Novo Extender PN−Blocks (ePN−Blocks) with Various Linkers To Construct Self-Assembling Chain-like Nanostructures. To construct oligomeric chainlike extended nanostructures, we designed de novo ePN−Blocks by tandemly fusing two de novo WA20 proteins with linkers of various type and length63,64 as shown in Figure 1 and Figure S1 (Supporting Information). The two WA20 domains were fused with helical linkers (HL: (EAAAK)n; n = 2−5; Figure 1B) or flexible linkers (FL: (GGGGS)n; n = 3, 4; Figure 1C). The helical linkers were derived from stable helix-forming de novo designed peptides described by Marqusee and Baldwin,65 and we previously demonstrated by using fluorescence resonance energy transfer and small-angle X-ray scattering (SAXS) techniques that the helical linkers can separate two domains of a fusion protein at controlled distances.63,64 Because WA20 forms a stable intermolecularly folded dimeric structure,23 ePN−Blocks were expected to selfassemble into cyclized chain-like homooligomers (Figure 1A), thereby giving rise to various nanostructures that depend on the different types and lengths of linkers. This notable series of PN−Blocks are completely “de novo proteins,” which have no sequences derived from any natural proteins. The de novo ePN−Block proteins were designed by tandemly linking the two de novo WA20 proteins, which were created from scratch using the binary code strategy,9,10 with artificial peptide linker sequences.63 Self-Assembling Homooligomers of ePN−Blocks. The present ePN−Block proteins with various linkers were expressed in Escherichia coli, and soluble and insoluble fractions of cells were prepared by centrifugation after disruption by sonication. SDS-PAGE analysis of the resulting fractions showed that ePN−Block proteins with long helical linkers (HL4, HL5) and flexible linkers (FL3, FL4) were expressed mainly in soluble fractions from E. coli lysates (Figure 2A). However, ePN−Block proteins with short helical linkers (HL2, HL3) were expressed mainly in insoluble fractions. After purification of soluble ePN−Block proteins using immobilized metal ion affinity chromatography (IMAC), SDS-PAGE showed a predominating single band at every ePN−Block lane (Figure 2B), whereas native PAGE showed migration ladders of ePN−Block proteins with the long helical linkers (HL4, HL5) and flexible linkers (FL3, FL4; Figure 2C). In contrast, ePN−Blocks with short helical linkers (HL2, HL3) migrated as a few bands in native PAGE (Figure 2C). These results suggest that ePN−Blocks with long helical linkers (HL4 and HL5) and flexible linkers (FL3 and FL4) form several stable homooligomeric states in soluble fractions from E. coli lysates. However, PN−Blocks with short helical linkers (HL2 and HL3) mainly precipitated in the insoluble fraction and formed only a few limited oligomeric states in the soluble fraction possibly because characteristics of short and rigid helical linkers (HL2 and HL3) may cause steric hindrances and lead to insoluble forms of ePN−Block homooligomers (Figure S2). Because insoluble samples are difficult to analyze using standard techniques for protein solutions, we performed further experiments on soluble samples of ePN−Blocks with HL4 and FL4, and compared these typical samples of helical and flexible

Figure 2. Polyacrylamide gel electrophoresis (PAGE) of ePN−Blocks with various linkers. (A) SDS-PAGE (17.5% polyacrylamide gel) of ePN−Blocks expressed in E. coli. M, protein molecular weight marker, broad (Takara Bio, Otsu, Japan); S, supernatant (soluble fraction); P, pellet (insoluble fraction); +, addition of 0.2 mM IPTG; −, no addition of IPTG. (B) SDS-PAGE (17.5% polyacrylamide gel) of ePN−Blocks and (C) native PAGE (5.0% polyacrylamide gel) of ePN−Blocks after immobilized metal ion affinity chromatography (IMAC) purification. W, stopper PN−Block (WA20).

linkers at the same linker length. After purification by IMAC followed by size exclusion chromatography (SEC) (Figures S3−S6), the fractionated samples of ePN−Block (HL4) and ePN−Block (FL4) homooligomers (Table S1) were analyzed by SEC-multiangle light scattering (SEC−MALS) and SAXS as described in more detail in the Supporting Information. In brief, the results of SEC−MALS are summarized in Table 1, Tables S2 and S3, and Figures S7 and S8, and the results of SAXS are summarized in Table S4 (Supporting Information), and Figure 3 and Figures S9 and S10, suggesting that ePN− Blocks form homoligomers from dimer to pentamer at least. The discussion is carried forward in the following part. Moreover, the SEC-fractioned samples of ePN−Block (HL4) homooligomers were observed by transmission electron microscopy (TEM) with negatively staining (Figure 4 and Figures S11−S14). Oligomeric states of the samples were previously analyzed by SEC−MALS and native PAGE (Figures S7 and S11, Supporting Information). TEM images (Figure 4A−D) show that the relatively larger size samples (TEM samples 61, 63, 65, and 67 including tetramer (e4), pentamer (e5), and higher oligomers) contained various spherical particles approximately 15−20 nm in diameter, roughly consistent with SAXS analyses. Numbers and sizes of observable particles gradually decreased with increasing sample numbers (corresponding to elution volume of SEC) and particles were rarely found in TEM sample 71 mainly including trimer (e3) (Figure 4E), suggesting that tetramer (e4) may be the observable limit, and trimer (e3) and dimer (e2) cannot be observed by TEM due to low molecular mass and small size. In 1383


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ACS Synthetic Biology Table 1. Summary of SEC−MALS Analyses PN−Block oligomers ePN−Block (HL4) homooligomer ePN−Block (HL4) homooligomer ePN−Block (HL4) homooligomer ePN−Block (HL4) homooligomer ePN−Block (HL4) homooligomer ePN−Block (FL4) homooligomer ePN−Block (FL4) homooligomer ePN−Block (FL4) homooligomer ePN−Block (FL4) homooligomer ePN−Block (FL4) homooligomer esPN−Block (HL4) heterocomplex esPN−Block (HL4) heterocomplex esPN−Block (HL4) heterocomplex esPN−Block (HL4) heterocomplex esPN−Block (FL4) heterocomplex esPN−Block (FL4) heterocomplex esPN−Block (FL4) heterocomplex esPN−Block (FL4) heterocomplex sPN−Block (WA20)

sample and peak Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Peak 1

(E), Peak 2 (D), Peak 1 (C), Peak 2 (B), Peak 1 (A), Peak 1 (E), Peak 2 (D), Peak 1 (C), Peak 1 (B), Peak 1 (A), Peak 1 (d), Peak 1 (b), Peak 1 (b), Peak 2 (b), Peak 3 (d), Peak 1 (c), Peak 1 (b), Peak 1 (b), Peak 2

molecular mass [kDa] 48.8 48.6 73.5 98.8 129 48.3 47.6 69.6 104 135 49.5 83.4 112 149 46.8 75.8 106 132 22.5

ePN−Block: sPN−Block 2:0 2:0 3:0 4:0 5:0 2:0 2:0 3:0 4:0 5:0 1:2 2:2 3:2 4:2 1:2 2:2 3:2 4:2 0:2

(e2) (e2) (e3) (e4) (e5) (e2) (e2) (e3) (e4) (e5) (e1s2) (e2s2) (e3s2) (e4s2) (e1s2) (e2s2) (e3s2) (e4s2) (s2)

native PAGE band (Figure S6 or Figure 6) Band Band Band Band Band Band Band Band Band Band Band Band Band Band Band Band Band Band

1 2 3 4 5 1 2 3 4 5 1 2 3 4 1 2 3 4

DLS data indicate that the size distribution significantly shifts to larger size with the addition of nickel ions probably because of the intermolecular assembly induced by the nickel ion coordination with several clusters of histidine residues on the surface of the WA20 structure (PDB code 3VJF)23 (Figure S16, Supporting Information). Negatively stained ePN−Block (FL4) homooligomer samples were also observed by TEM (Figures S17−S19, Supporting Information). TEM images show various shape nanoparticles and aggregations. Numbers and sizes of observable particles gradually decreased with increasing sample numbers corresponding to elution volume of SEC, as described in more detail in the Supporting Information. Reconstruction of Heterooligomeric Extender and Stopper PN−Block Complexes by Denaturation and Refolding. To expand the possibilities of the PN−Block strategy, we reconstructed multicomponent PN−Block complexes from extender PN−Block (ePN−Block) and stopper PN−Block (sPN−Block, i.e., WA20 protein) by denaturation and refolding. In analogy with supramolecular chemistry,57 the chain length of supramolecular polymers can be tuned by the addition of a monofunctional monomer (sPN−Block). The chain stopper can be expected to limit the growth of selfassembling bifunctional monomers (ePN−Blocks) and lead to altering conformation and reduce polydispersity. Figure 5A shows native PAGE analysis following reconstruction of ePN− Block (HL4) and sPN−Block (WA20) proteins. Before denaturation, band patterns of ePN−Block (HL4) did not change in just mixed samples with sPN−Block. In contrast, after denaturation and refolding, new pattern bands (Figure 4A, black arrowheads) and diminished bands (Figure 5A, gray arrowheads) appeared with increasing sPN−Block contents, suggesting the formation of several heterooligomeric complexes of ePN−Blocks and sPN−Blocks (esPN−Block heterocomplexes). After denaturation and refolding of ePN−Block (FL4) and sPN−Block proteins, a stronger band and several weaker bands (Figure 5B, black and gray arrowheads, respectively) were seen with increasing sPN−Block contents, suggesting formation of heterooligomeric esPN−Block complexes from ePN−Block (FL4) and sPN−Block proteins. These results

Figure 3. Small-angle X-ray scattering (SAXS) analyses of the ePN− Block homooligomer samples. Concentration-normalized absolute scattering intensities I(q)/c of ePN−Block (HL4) [e(HL4)] homooligomer samples (A) and ePN−Block (FL4) [e(FL4)] homooligomer samples (B). Chicken egg lysozyme was used as a molecular mass reference standard. Their real-space information, concentration-normalized pair-distance distribution functions p(r)/c of ePN−Block (HL4) homooligomer samples (C) and ePN−Block (FL4) homooligomer samples (D), which were calculated using indirect Fourier transformation (IFT).

contrast, the TEM image of sample 71 with the addition of nickel ions shows many large aggregations as nanoscale spotted patterns (Figure 4F), suggesting further supramolecular assembly of ePN−Block (HL4) homooligomers induced by nickel ions. The addition of nickel ions can promote intermolecular interactions between WA20 domains, as suggested by dynamic light scattering (DLS) experiments of WA20 proteins with and without nickel ions (Figure S15). The 1384


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Figure 4. TEM imaging of SEC-fractioned and negatively stained ePN−Block (HL4) homooligomers. (A) TEM image of ePN−Block (HL4) sample 61 mainly including tetramer, pentamer (major), and higher oligomers. (B) TEM image of ePN−Block (HL4) sample 63 mainly including tetramer and pentamer. (C) TEM image of ePN− Block (HL4) sample 65 mainly including tetramer (major) and pentamer. (D) TEM image of ePN−Block (HL4) sample 67 mainly including tetramer (major) and trimer. (E) TEM image of ePN−Block (HL4) sample 71 mainly including trimer. (F) TEM image of ePN− Block (HL4) sample 71 with the addition of 5 μM NiCl2. These fullsize images are shown in Figure S12. Oligomeric states of these samples were analyzed by SEC−MALS and native PAGE (Figures S7 and S11).

Figure 5. Reconstruction of heterooligomeric complexes from multicomponent extender and stopper PN−Blocks by denaturation and refolding. Native PAGE (7.5% polyacrylamide gel) analysis of the reconstruction of extender PN−Block (ePN-block) and stopper PN− Block (sPN−Block): (A) ePN-block (HL4) and (B) ePN-block (FL4). In the left half, samples were just mixed (Mixture), and in the right half samples were denatured and refolded after mixing (Reconstruction). The sPN-block (WA20) was added at stepwise increases in the ratio of sPN−Block/ePN−Block of 1, 2, 4, and 8. (C) Schematics of the reconstruction process from ePN−Blocks (red and blue) and sPN−Blocks (light gray) to esPN−Block heterocomplexes.

(HL4 or FL4) in a wide range of fractions across higher molecular size complexes. In subsequent SAXS analyses, samples were derived from several SEC fractions of esPN−Block heterocomplexes (Figure 6, Figure S24, and Table S5). Some samples were selected for SEC−MALS analysis and the results are summarized in Table 1 and Figures S25 and S26 (Supporting Information). Molecular masses of esPN−Block heterocomplexes reveal the presence of several species of the esPN−Block heterocomplexes containing one ePN−Block and two sPN−Blocks (e1s2), two ePN− Blocks and two sPN−Blocks (e2s2), three ePN−Blocks and two sPN−Blocks (e3s2), and four ePN−Blocks and two sPN− Blocks (e4s2), as indicated in native PAGE analyses (Figure 6, bands 1, 2, 3, and 4, respectively). Figure 7A and B show SAXS intensities of esPN−Block heterocomplex and sPN−Block (WA20) samples with chicken egg lysozyme as a molecular mass reference standard (Mw, 14.3 kDa). Assuming that these proteins have practically identical scattering length densities and specific volumes, and the structure factor S(q) ≈ 1 for screened electrostatic repulsion

imply that several esPN−Block complexes of extended open chain-like heterooligomers were reconstructed after denaturation and refolding of multicomponent ePN−Blocks and sPN− Blocks (Figure 5C). Oligomeric State Analyses of esPN−Block Heterocomplexes. To investigate the oligomeric states of esPN− Block heterocomplexes, we fractionated esPN−Blocks before and after reconstruction using SEC on a Superdex 200 increase 10/300 GL column (Figures S20−S23). Before reconstruction (Figures S20 and S22), SEC chromatograms and SDS-PAGE analyses indicated that ePN−Block (HL4 or FL4) and sPN− Block (WA20) proteins had not formed hetero-oligomeric complexes because the sPN−Block was eluted in only low molecular size fractions that corresponded with those of the WA20 homodimer. In contrast, reconstruction by denaturation and refolding (Figures S21 and S23, Supporting Information) clearly led to the formation of hetero-oligomeric esPN−Block complexes from ePN−Block (HL4 or FL4) and sPN−Block (WA20) because sPN−Block was eluted with ePN−Block 1385


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Figure 6. Native PAGE analysis of the esPN−Block heterocomplex samples (Table S5) separated by size exclusion chromatography (SEC). (A) Native PAGE (7.5% polyacrylamide gel) of esPN−Block (HL4) heterocomplexes; Q, mixed sample of ePN−Block and sPN− Block before reconstruction and SEC separation; R, esPN−Block heterocomplex sample after reconstruction before SEC separation; W, sPN−Block (WA20). (B) Schematics of the esPN−Block (HL4) heterocomplexes. (C) native PAGE (7.5% polyacrylamide gel) of esPN−Block (FL4) heterocomplexes. (D) schematics of esPN−Block (FL4) heterocomplexes. The same colors are used in Figures 1 and 4. Theoretical molecular masses of esPN−Block heterocomplexes are presented in parentheses. SDS-PAGE analyses of these samples are also shown in Figure S24.

in dilute samples, the forward scattering intensity normalized by protein concentration I(q → 0)/c is proportional to the weightaverage molecular mass (Mw). Mw of these samples (Table 2) is roughly consistent with SEC−MALS analysis, when sample purity of complex components (Figure 6 and Table 2) and experimental errors are considered. Shape Analyses of esPN−Block Heterocomplexes. To calculate intuitive real-space data from SAXS analyses, pairdistance distribution functions p(r) were determined using indirect Fourier transformation,66−68 which were reflected by shapes and components of the esPN−Block heterocomplex samples (Figure 7C,D), while the complex samples had some polydispersity derived from equilibria of cyclized/extended chain forms in the reconstruction process. The p(r) series of the esPN−Block (HL4) heterocomplex samples (Figure 7C) was characterized as an extended tail in the high-r regime, suggesting that the esPN−Block (HL4) heterocomplexes form extended shapes. In contrast, the p(r) series of the esPN−Block (FL4) heterocomplexes (Figure 7D) had shorter Dmax values than those of the esPN−Block (HL4) heterocomplexes, implying that the esPN−Block (FL4) heterocomplexes form relatively more compact shapes than the esPN−Block (HL4) heterocomplexes. In comparison of p(r) of ePN−Block homooligomers with those of esPN−Block heterocomplexes (Figure S27), the Dmax values of the esPN−Block (HL4)

Figure 7. SAXS analyses of the esPN−Block heterocomplex samples. Concentration-normalized absolute scattering intensities I(q)/c of esPN−Block (HL4) heterocomplex [es(HL4)] samples (A) and esPN−Block (FL4) [es(FL4)] heterocomplex samples (B). WA20 was used as a control and chicken egg lysozyme was used as a molecular mass reference standard. The real-space functions, concentrationnormalized pair-distance distribution functions p(r)/c of the esPN− Block (HL4) heterocomplex samples (C) and the esPN−Block (FL4) heterocomplex samples (D). Dimensionless Kratky plots of the esPN− Block (HL4) heterocomplex samples (E) and the esPN−Block (FL4) heterocomplex samples (F) (0 < q < 3 nm−1).

heterocomplex samples tend to be longer than those of the ePN−Block (HL4) homooligomer samples while the samples had polydispersity. In addition, the p(r) series of ePN−Block (FL4) homoligomers and esPN−Block (FL4) heterocomplexes shows relatively smaller differences in shapes possibly due to flexible characteristics of the flexible linker (FL4). Kratky plots and dimensionless Kratky plots of esPN−Block heterocomplex samples are shown in Figure S28 (Supporting Information) and Figure 7E,F. These plots are useful representations of the SAXS intensity to quickly assess the globular nature of a polypeptide chain without any modeling.69 In dimensionless Kratky plots normalized by I(0) and Rg, the 1386


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ACS Synthetic Biology Table 2. Summary of SAXS Analyses of esPN−Block Heterocomplexes

a

esPN−Block heterocomplex samples

I(q→0)/c [cm−1mg−1mL]

Rg [nm]

Dmax [nm]

Mw [kDa]

components of esPN−Block heterocomplexesa

es(HL4) sample (a) es(HL4) sample (b) es(HL4) sample (c) es(HL4) sample (d) es(HL4) sample (e) es(FL4) sample (a) es(FL4) sample (b) es(FL4) sample (c) es(FL4) sample (d) es(FL4) sample (e) sPN−Block (WA20) Lysozyme

0.133 0.0760 0.0619 0.0359 0.0353 0.102 0.0712 0.0401 0.0310 0.0248 0.0152 0.00870

11.0 8.6 6.9 4.7 3.6 8.8 6.4 4.8 3.6 3.3 2.6 1.5

52 40 29 20 15 35 27 20 15 12 10 4.5

219 125 102 59.0 58.1 167 117 65.9 51.0 40.8 25.0 14.3

e3s2, e4s2, higher e2s2, e3s2, e4s2, higher e2s2, e3s2 e1s2, e2s2 e1s2 e4s2, higher e3s2, e4s2, higher e2s2, e3s2 e1s2, e2s2 e1s2 s2

Main components are indicated by boldface. These components were judged mainly by the SEC−MALS and native PAGE results.

Figure 8. Three-dimensional structural modeling of the esPN−Block (HL4) heterocomplex based on SAXS analysis. (A) A rigid-body model structure of one extender and two stoppers (e1s2) of the esPN−Block (HL4) heterocomplex [e1s2(HL4)]. The model is shown as a ribbon representation in the translucent surface representation. The first WA20 domain (red), the helical linker (yellow), and the second WA20 domain (blue) of ePN−Block; sPN−Block (light gray). (B) The concentration-normalized scattering intensity of the esPN−Block (HL4) heterocomplex sample (e) obtained by the SAXS experiment (open circle) and fitting of I(q) simulated from the rigid-body model of e1s2 (HL4) (red line). (C) The pair-distance distribution function p(r) of the esPN−Block (HL4) heterocomplex sample (e) calculated from the SAXS data (black dash line) and p(r) simulated from the rigid-body model of e1s2 (HL4) (red line). (D) A dummy atom model of e1s2(HL4) reconstructed from the SAXS data using ab initio modeling programs DAMMIF, DAMAVER, and DAMMIN. (E) The concentration-normalized scattering intensity of the esPN− Block (HL4) heterocomplex sample (e) (black open circle) and fitting of I(q) simulated from the DAMMIN model of e1s2(HL4) (red line). (F) Superimposition of the rigid-body model (magenta ribbon representation) and the dummy atom model (green) of e1s2 (HL4). Blue dots represent an averaged model from 10 structural models calculated by DAMMIF and DAMAVER (Figure S31).

information about size of the protein is removed but the information about the shape and flexibility is kept.70 The dimensionless Kratky plot of lysozyme (a reference sample) exhibits a bell-shape with a well-defined peak at a maximum value of 1.104 for qRg = √3, indicating a typical globular protein shape. In the case of the dimensionless Kratky plots of the esPN−Block (HL4) heterocomplexes (Figure 7E), the peaks of the bell-shapes tend to shift to the upper right in larger complexes, suggesting that the larger complexes have the more extended shapes as simulated in Figure S29A. In addition, the dimensionless Kratky plots of the esPN−Block (FL4) heterocomplex samples have a gentler hill-like broad peak

than those of esPN−Block (HL4) heterocomplex samples, implying that the esPN−Block (FL4) heterocomplexes had elongated shapes and more flexible conformation of multidomains, such as flexible and extended conformation proteins with some domains tethered by linkers69 (e.g., calmodulin,71 Filamin C 23−24,72 p47phox73 and p67phox74) (Figure S29A and B), than the esPN−Block (HL4) heterocomplexes because of distinctive features of the different linkers. However, the polydispersity of the samples may be not negligible and complicate the interpretation of these results. Moreover, the dimensionless Kratky plots of comparable samples of the ePN−Block homooligomer sample (C) and 1387


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Figure 9. Three-dimensional structural modeling of the esPN−Block (FL4) heterocomplex based on SAXS analysis. (A) Rigid-body model structures of one extender and two stoppers (e1s2) of the esPN−Block (FL4) heterocomplex [e1s2 (FL4)]. The models are shown as ribbon representation in translucent surface representation. The first WA20 domain (red), the flexible linker (black), and the second WA20 domain (blue) of ePN−Block; sPN−Block (light gray). (B) The concentration-normalized scattering intensity of the esPN−Block (FL4) heterocomplex sample (e) obtained by the SAXS experiment (open circle) and fitting of I(q) simulated from the two rigid-body model structures of e1s2 (C form:V form = 1:1) (red line). (C) The pair-distance distribution function p(r) of the esPN−Block (FL4) heterocomplex sample (e) calculated from the SAXS data (black dash line) and p(r) simulated from the two rigid-body model structures of e1s2 (C form:V form = 1:1) (red line). (D) A dummy atom model of e1s2 (FL4) reconstructed from the SAXS data using ab initio modeling programs, DAMMIF, DAMAVER, and DAMMIN. (E) The concentrationnormalized scattering intensity of the esPN−Block (FL4) heterocomplex sample (e) (black open circle) and fitting of I(q) simulated from the DAMMIN model of e1s2 (FL4) (red line). (F) Superimposition of the rigid-body models (ribbon representations of yellow C form and magenta V form) and the dummy atom model (green) of e1s2 (FL4). Blue dots represents an averaged model from 10 structural models calculated by DAMMIF and DAMAVER (Figure S34).

extended “Z” shape (Figure 8A), and the simulated I(q) and p(r) from the rigid-body model closely resembles that from the SAXS experiment (Figure 8B,C). Moreover, the low-resolution dummy atom models of e1s2 (HL4) were constructed from the SAXS data using ab initio shape modeling programs DAMMIF,75 DAMAVER,76 and DAMMIN77 (Figure 8D,E and Figure S31). Ten times independent calculations of ab initio shape determination by DAMMIF reproducibly generated typical elongated shape models (Figure S31A). The refined DAMMIN model of e1s2 (HL4) is superimposed on the rigid body model of e1s2 (HL4) as shown in Figure 8F. This Z-like shape conformation of e1s2 (HL4) resembles the conformation of chimeric proteins of green fluorescent protein variants diagonally linked by the helical linkers previously analyzed by SAXS.64 In addition, we also tried to analyze shapes of the esPN− Block heterocomplex (HL4) sample (c) mainly containing e2s2 and e3s2 complexes judged from the native PAGE and SEC− MALS data (Figure 6 and Table 1 and Figure S25). On the basis of the p(r) profiles and Dmax values (Figure 7C), we speculate that the possible structural models of e2s2 (HL4) and e3s2 (HL4) may be constructed by a chain-like extension of e1s2 (HL4) model as a basic structural unit, as shown in the schematics in Figure 6B and Figure S29A. However, it is difficult to construct reliable models of e2s2 and e3s2 from the SAXS data due to polydispersity of the samples.

esPN−Block heterocomplex sample (c) mainly containing homooligomers (e3, e4) or heterocomplexes (e2s2, e3e4) are shown in Figure S30. The plots of the ePN−Block (FL4 and HL4) homooligomer (C) and esPN−Block (FL4) heterocomplex (c) have a gentle hill-like broad peak, suggesting their flexible and dynamic conformation in contrast to a globular protein (e.g., lysozyme) and the esPN−Block (HL4) heterocomplex samples. By contrast, the esPN−Block (HL4) heterocomplex samples have a distinctive feature of a big bellshape peak, suggesting that they are well-folded proteins with large Rg (i.e., elongated conformations with some rigidity) as simulated in Figure S29A. Also noteworthy is that the plots of the ePN−Block (HL4) homooligomer (C) and the esPN− Block (HL4) heterocomplex sample (c) represent significantly different features, implying that topology and shape were transformed from cyclized chain-like closed forms of the ePN− Block homoligomers into extended chain-like open forms of the heterooligomeric esPN−Block complexes by the reconstruction process as shown in the schematics (Figure 5C). Since the esPN−Block (HL4) heterocomplex sample (e) contained predominantly the e1s2 (HL4) complex in native PAGE analysis (Figure 6A), for further structural analysis, a rigid-body model structure of e1s2 (HL4) was constructed based on the crystal structure of the WA20 dimer (PDB code 3VJF)23 to explain the experimental p(r) with consideration of the helical linker rigidity. The model of e1s2 (HL4) shows an 1388


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ACS Synthetic Biology Rigid-body model structures of the esPN−Block heterocomplex e1s2 (FL4) were also constructed based on the WA20 crystal structure. The “V” form of a rigid-body model for e1s2 (FL4) was built in terms of the Dmax value (∼12 nm) (Figure 9A). The p(r) value that was calculated from the V form model only poorly resembled that from the SAXS experiment (Figure S32, Supporting Information). Thus, an additional rigid-body model of a compact form (C form) was constructed with consideration of linker flexibility and domain interactions. Subsequently, the I(q) and p(r) of the esPN−Block (FL4) heterocomplex sample (e), which comprised mainly the e1s1 (FL4) heterocomplex, was simulated well with the composite I(q) and p(r) of an equal ratio of V and C form models (Figure 9B,C). These observations imply that dynamic structures of e1s2 (FL4) represent structural ensembles including the V form, the C form, and many transient intermediates. Therefore, we tried multistate modeling with SAXS profiles using MultiFoXS server78 as described in the Supporting Information. The summarized results shown in Figure S33 also suggest that the e1s2 (FL4) heterocomplex are conformationally heterogeneous and dynamic. In addition, the low-resolution dummy atom models of e1s2 (FL4) were constructed from the SAXS data using the ab initio shape modeling programs (Figure 9D,E and Figure S34). Ten times calculations of ab initio shape determination by DAMMIF generated various structural models (Figure S34A), also suggesting that the e1s2 (FL4) conformation is more heterogeneous and dynamic than e1s2 (HL4). The rigid-body models (V and C forms) of e1s2 (FL4) are roughly superimposed on the DAMMIN model of e1s2 (FL4) as shown in Figure 9F. Atomic Force Microscopy (AFM) Observations of SelfAssembling Supramolecular Nanostructures of esPN− Block Complexes. To expand the potential of PN−Block strategy for nanotechnology, esPN−Block heterocomplexes further self-assembled into supramolecular nanostructures with metal ions (Ni2+). As suggested by the TEM experiment (Figure 4E,F) and the DLS experiment (Figure S15), the addition of nickel ions can promote intermolecular interactions between the WA20 domains of PN−Blocks. The esPN−Block heterocomplexes with nickel ions were observed in liquid using frequency modulation (FM)-atomic force microscopy (AFM) (Figure 10 and Figures S35−S37). The resulting image (Figure 10A) shows self-assembling supramolecular nanostructures of the esPN−Block (HL4) heterocomplex fraction 20 (Figure S21, Supporting Information), comprising mainly e2s2 (HL4) and e3s2 (HL4) heterocomplexes. Moreover, many bundles of rodlike structures of 10.9 ± 1.8 nm in length and 3.4 ± 1.8 nm in width were found in the AFM image (Table S6, Supporting Information). The sizes of the rodlike structural domains are consistent with the size of ∼10 nm in length and ∼3 nm in width estimated from the crystal structure (PDB code 3VJF)23 of the WA20 dimer (Figure S16, Supporting Information). Several domains of WA20 units seem to be aligned laterally in the direction of the longitudinal axis (Figure S35A and B). In contrast, Figure 10B shows an AFM image of selfassembling nanostructures of the esPN−Block (FL4) heterocomplex fraction 19 comprising e2s2 (FL4), e3s2 (FL4), and e4s2 (FL4) (Figure S23). The stripe features in Figure 10B could be AFM scan artifacts as they are mostly oriented in the slow scan direction. However, similar nanostructures were reproducibly observed in the images obtained with different scan sizes, and more magnified images indicate that their orientations are not necessarily the same (Figure S36). Thus,

Figure 10. Atomic force microscopy (AFM) imaging of selfassembling supramolecular nanostructures of the esPN−Block heterocomplexes with metal ions (Ni2+) on mica surfaces in liquid. (A) AFM image of the esPN−Block (HL4) heterocomplex fraction 20 (Figure S21, Supporting Information) comprising mainly e2s2 (HL4) and e3s2 (HL4). (B) AFM image of the esPN−Block (FL4) heterocomplex fraction 19 (Figure S23) comprising e2s2 (FL4), e3s2 (FL4), and e4s2 (FL4). The additional images obtained with different scan sizes and more magnified images are shown in Figure S36.

these strip features are not due to the scan artifacts but represent the true surface structures. A number of bundles of rodlike structures with lengths of 10.2 ± 2.0 nm and widths of 3.2 ± 0.4 nm (Table S6) represented the WA20 structural domains, and these rodlike structural domains seem to line up and extend in the direction of the lateral axis (Figure S35C and D). These contrasting observations may reflect different structural properties of esPN−Block complexes, including differences in rigidity and flexibility of helical (HL4) and flexible (FL4) linkers.

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CONCLUSIONS We designed and created fully de novo ePN−Block proteins by tandemly fusing the two de novo WA20 proteins with various linkers, as a new series of PN−Blocks. Analyses of ePN−Blocks by native PAGE, TEM, SEC−MALS, and SAXS indicate the formation of several homooligomeric states. Then, we reconstructed heterooligomeric esPN−Block complexes by denaturating and refolding ePN−Blocks and sPN−Blocks. The series of comprehensive SEC−MALS and SAXS analyses suggested that the cyclized chain-like ePN−Block homooligomers were transformed into different types of extended chain-like structures of esPN−Block heterocomplexes depending on their linker type, demonstrating great potential of reconstructible PN−Blocks as artificial nanobuilding block molecules. Moreover, AFM observations revealed that the esPN−Block heterocomplexes further self-assembled into higher-order supramolecular nanostructures with metal ions. These results demonstrate that the PN−Block strategy is a useful and systematic strategy for constructing novel nanoarchitectures of de novo protein-based supramolecular polymer complexes for potential nanobiomaterials in biotechnology and synthetic biology.

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METHODS Construction of Protein Expression Plasmids. A DNA fragment encoding the de novo protein WA20 was prepared from the plasmid pET3a-WA2023 using polymerase chain reactions (PCR) with KOD-Plus-Neo DNA polymerase 1389


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(pH 7.5) containing 100 mM NaCl, 10% glycerol, and 200 mM L-arginine hydrochloride (ArgHCl) using a Bio-Tech oscillatory microdialysis system (BM Equipment, Tokyo, Japan). Concentrated samples of refolded ePN−Block and sPN−Block heterocomplexes (esPN−Blocks) were separated using size exclusion chromatography (SEC), and were eluted in 20 mM HEPES buffer (pH 7.5) containing 100 mM NaCl, 10% glycerol, and 200 mM ArgHCl from a Superdex 200 Increase 10/300 GL column (GE healthcare, Little Chalfont Buckinghamshire, UK). Size Exclusion Chromatography−Multiangle Light Scattering (SEC−MALS). SEC−MALS experiments were performed using a 1260 Infinity HPLC system (Agilent Technologies, Santa Clara, CA) equipped with a Superdex 200 Increase 10/300 GL column, which was connected in line with a miniDAWN TREOS multiangle static light scattering detector (Wyatt Technology, Santa Barbara, CA). Data were collected at 20 °C in phosphate buffered saline (PBS, pH 7.4) comprising 1 mM KH2PO4, 3 mM Na2HPO4, and 155 mM NaCl, and were analyzed using ASTRA 6 software (Wyatt Technology). A dn/dc value of 0.185 mL/g was generally used for proteins, with extinction coefficients of 0.507 and 0.519 mL mg−1 cm−1 for ePN−Block/esPN−Block (HL4) and ePN− Block/esPN−Block (FL4), respectively, as calculated according to amino acid sequences. Small-Angle X-ray Scattering (SAXS). SAXS measurements were performed on several fraction samples (Table S1, Supporting Information) of ePN−Block homooligomers and esPN−Block heterocomplexes after separation by SEC, WA20 (sPN−Block), and chicken egg white lysozyme (Wako Pure Chemical Industries, Osaka, Japan) in 20 mM HEPES buffer (pH 7.5) containing 100 mM NaCl, 200 mM ArgHCl, and 10% glycerol at 20 °C using synchrotron radiation (λ = 0.1488 nm) with a PILATUS3 2 M detector (Dectris, Baden, Switzerland) at the Photon Factory BL-10C beamline (KEK, Tsukuba, Japan).79 Two-dimensional scattering images were integrated into one-dimensional scattering intensities (I(q)) as a function of the magnitude of the scattering vector q = (4π/λ) sin(θ/2) using the FIT2D program,80 where θ is the total scattering angle. The scattering intensity for a colloidal dispersion is generally given by the product of the form factor P(q) and structure factor S(q). Hence, I(q) = nP(q) S(q), where n is the number density of the particle. In the present experiments, the structure factor was almost at unity (I(q) ≈ nP(q)), because interparticle interactions such as the excluded volume effect and electrostatic interactions can be neglected at low protein and high salt concentrations. Thus, the form factor is given by the Fourier transformation of the pair-distance distribution function p(r), which expresses the size and shape of the particle as follows:

(Toyobo, Osaka, Japan) and primers for the T7 promoter primer and WA20RV_HindIII (Table S7, Supporting Information). The amplified fragment was digested using NdeI and HindIII and was cloned into pET32/EBFP-HL5-EGFP63 between the NdeI and HindIII sites to construct the plasmid pET-WA20-HL5-GFP, in which the Trx tag and the EBFP gene were replaced with the WA20 gene. Another DNA fragment encoding WA20 was prepared from the plasmid pET3a-WA20 using PCR with primers for WA20FW_NotI and WA20RV_XhoI (Table S7). The amplified fragment was then digested using NotI and XhoI and was cloned into pET-WA20-HL5EGFP between the NotI and XhoI sites to give the expression plasmid pET-WA20-HL5-WA20 for an extender PN−Block (ePN−Block). DNA fragments encoding the other linker genes (HL2, HL3, HL4, FL3, and FL4) were prepared by digestion of the plasmid pET32/EBFP-linker-EGFP63 with HindIII and NotI and cloning of fragments into pET-WA20-HL5-WA20 between the restriction sites to give the plasmids pET-WA20HL2-WA20, pET-WA20-HL3-WA20, pET-WA20-HL4-WA20, pET-WA20-FL3-WA20, and pET-WA20-FL4-WA20, respectively. The amino acid sequences of the ePN−Block proteins are shown in Figure S1. Protein Expression and Purification. All ePN−Block proteins were expressed in E. coli BL21 Star(DE3) (Invitrogen, Carlsbad, CA) harboring pET-WA20-Linker-WA20 in 2 L of LB broth, Lennox (Nacalai Tesque, Kyoto, Japan) containing 50 μg/mL ampicillin sodium salt at 37 °C. Protein expression was induced using 0.2 mM β-D-1-thiogalactopyranoside at an optical density OD600 of about 0.8 at 600 nm, and cells were further cultured for 3−4 h at 37 °C. Proteins were extracted from harvested cells by sonication in lysis buffer containing 50 mM sodium phosphate buffer (pH 7.0), 300 mM NaCl, and 10% glycerol. Proteins were then purified using IMAC with TALON metal affinity resin (Clontech, Takara Bio, Mountain View, CA) according to the manufacturer’s protocols. The equilibration/wash buffer contained 50 mM sodium phosphate buffer (pH 7.0) and 300 mM NaCl, and the elution buffer contained 50 mM sodium phosphate buffer (pH 7.0), 300 mM NaCl, 10% glycerol, and 250 mM imidazole. Since many histidine residues are exposed on the surface of the WA20 structure (Figure S16),23 WA20 and ePN−Block proteins can bind TALON metal affinity resin even without a His-tag. Protein expression and IMAC purification of the stopper PN− Block (sPN−Block; WA20) were performed as described previously.23 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analyses were performed according to the standard Laemmli procedure. The stacking gel contained 4.5% polyacrylamide, 125 mM Tris−HCl (pH 6.8), and 0.1% SDS, and the separating gel comprised 17.5% polyacrylamide, 125 mM Tris−HCl (pH 8.8). Proteins were electrophoresed in running buffer containing 25 mM Tris, 192 mM glycine, and 0.1% SDS. For native PAGE analyses, stacking gels, separating gels (5% or 7.5% polyacrylamide), and running buffer were prepared as for SDS-PAGE, but with no SDS. Proteins in the gels were stained with Coomassie brilliant blue (CBB) R-250. Denaturation, Refolding, and Further Purification of PN−Block Proteins. The ePN−Block (HL4 or FL4) protein was mixed with the sPN−Block protein (WA20), and they were denatured in 6 M guanidine hydrochloride (GdnHCl) for 3 h at 25 °C in 20 mM HEPES buffer (pH 7.5) containing 100 mM NaCl and 10% glycerol. For refolding, denatured proteins were dialyzed three times for about 4 h against 20 mM HEPES buffer

P(q) = 4π

∫0

Dmax

p(r )

sin qr dr qr

where Dmax is the maximum intraparticle distance. The indirect Fourier transformation (IFT) technique was used to calculate p(r) for particles with a virtually model-free routine.66−68 Forward scattering intensity I(q→0) was extrapolated from SAXS data, and the radius of gyration Rg, was estimated using the Guinier approximation.67 Modeling Analyses. Rigid-body models of esPN−Block heterocomplexes were constructed using the program Coot81 and Foldit Standalone82 based on the crystal structure of the de 1390


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ACS Synthetic Biology novo protein WA20 dimer (protein data bank (PDB) code, 3VJF)23 with consideration of N- and C-terminal directions. Rigid-body models were manually and iteratively refined to minimize differences between that calculated p(r) from models and those determined in SAXS experiments. The program CRYSOL83 in the ATSAS program suite84 was used for evaluating the solution scattering from the models and fitting it to experimental scattering curves with the χ2 value.71 The lowresolution dummy atom models were constructed from the SAXS data using ab initio shape modeling programs as follows (Figures S31 and S34). Calculations of rapid ab initio shape determination were performed 10 times by DAMMIF75 without a symmetry constraint, and the generated models were aligned and averaged by DAMAVER76 in the ATSAS program suite84 for small-angle scattering data analysis from biological macromolecules. The DAMAVER model was modified with fixed core by DAMSTART and further refinement of the model was performed by DAMMIN.77 Superimposing model structures were performed by SUPCOMB.85 In addition, multistate structural modeling of the esPN−Block (FL4) heterocomplex e1s2 (FL4) was performed based on SAXS profiles using MultiFoXS server78 as described in more detail in the Supporting Information. The SAXS data of the esPN−Block (HL4 and FL4) heterocomplex samples (e) and the rigid-body and dummy-atom models of e1s2 (HL4) and e1s2 (FL4) have been deposited into the Small Angle Scattering Biological Data Bank86 (SASBDB accession codes: SASDD46 for e1s2 (HL4), SASDD56 for e1s2 (FL4)). Transmission Electron Microscopy (TEM) Imaging. After IMAC purification, concentrated ePN−Block homooligomer samples were fractionated in 20 mM HEPES buffer (pH 7.5) containing 100 mM NaCl, 10% glycerol, and 200 mM ArgHCl using SEC on a HiLoad 16/600 Superdex 200 pg (GE healthcare) column (Figures S11, S13, and S17). The samples (∼0.1 mg/mL protein concentration) were negatively stained with 2%(w/v) uranyl acetate on carbon-supported copper grids glow-discharged beforehand, and then imaged by transmission electron microscopy (TEM) with a 200 kV field-emission electron source and a zero-loss imaging of omega-type energy filter (JEM2200FS, JEOL, Tokyo, Japan). Atomic Force Microscopy (AFM) Imaging. Solutions of 5 mM NiCl2 were deposited onto freshly cleaved mica surfaces (ϕ12 mm). After 5 min, surfaces were gently rinsed 10 times in superpure water and were then dried by blowing with nitrogen gas. Samples of esPN−Block heterocomplexes were diluted in 10 mM PBS buffer to a concentration of about 10 μg/mL. Prior to frequency modulation atomic force microscopy (FM-AFM) imaging, esPN−Block complex sample solutions (100 μL) were deposited onto nickel ion-coated mica surfaces. Samples were then incubated for 5 min at room temperature (25 °C). Selfassembled nanostructures of esPN−Block (HL4) heterocomplex or esPN−Block (FL4) heterocomplex on mica surfaces were then rinsed with PBS. AFM measurements were performed using an in-house-built, ultralow-noise FM atomic force microscope87 combined with a commercially available AFM controller (ARC2, Asylum Research, Santa Barbara, CA). All AFM experiments were performed at room temperature (25 °C) in PBS buffer solution using a silicon cantilever (PPPNCH, NanoWorld, Neuchâtel, Switzerland) with a nominal spring constant of 42 N m−1 and a resonance frequency of 150 kHz in liquid. A phase-locked loop circuit (Nanonis OC4, SPECS Zurich, Zurich, Switzerland) was used to detect frequency shifts and to oscillate the cantilever with a constant

amplitude at its resonance frequency. Sizes of rod-like structural domains in AFM images were measured using the ImageJ program.88

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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/acssynbio.8b00007.

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Supplementary text, Tables S1−S7, Figures S1−S37, and related descriptions (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Takaaki Sato: 0000-0002-3455-4349 Takeshi Fukuma: 0000-0001-8971-6002 Ryoichi Arai: 0000-0001-5606-8483 Author Contributions

N.K. and R.A. designed the research; M.H.H. created the de novo protein WA20; N.K., K.I. and K.S. performed protein expression and purification experiments; N.K., K.I., T.S., and R.A performed SAXS experiment and analysis; N.K., K.M., and T.F. performed AFM experiment and analysis; C.S., N.K. and K.M. performed TEM experiment and analysis; N.K. and R.A. wrote the manuscript; and all authors discussed results, commented on the manuscript, and revised it. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Nobuyasu Koga, Dr. Rie Koga, and Dr. Takahiro Kosugi at the Institute for Molecular Science (IMS) for assistance in SEC−MALS experiments. We are grateful to Prof. Teruyuki Nagamune at University of Tokyo for kindly providing the plasmids pET32/EBFP-linker-EGFP. We also thank Prof. Nobuaki Hayashida at Shinshu University and Dr. Shinya Honda at Advanced Industrial Science and Technology for helpful advice. This work was supported by Joint Research of IMS and the Bio-AFM summer school at Kanazawa University. We thank Dr. Nobutaka Shimizu, Dr. Noriyuki Igarashi, and Photon Factory (PF) staff for their help in synchrotron SAXS experiments, which were performed at PF, KEK, under the approval of PF program advisory committee (Proposal Nos. 2014G111, 2016G153, and 2016G606). We are indebted to Divisions of Gene Research and Instrumental Analysis of Research Center for Supports to Advanced Science, Shinshu University, for providing facilities. This work was supported by JSPS Research Fellowships (DC2) and JSPS KAKENHI Grant Nos. JP14J10185 and JP16H06837 to N.K., and JSPS KAKENHI Grant Nos. JP22113508, JP24113707 (Innovative Areas “Intrinsically Disordered Proteins”), JP24780097, JP26288101, JP16K05841, JP16H00761 (Innovative Areas “Dynamical Ordering & Integrated Functions”), JSPS Postdoctoral Fellowships for Research Abroad, and Program for Dissemination of Tenure-Track System, to R.A. The work was also supported by NSF Grants MCB-1050510 and MCB-1409402 to M.H.H. 1391


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Self-Assembling Supramolecular Nanostructures Constructed from de

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