Designed Two- and Three-Dimensional Protein Nanocage Networks

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Designed 2D and 3D Protein Nanocage Networks Driven by Hydrophobic Interactions Contributed by Amyloidogenic Motifs Bowen Zheng, Kai Zhou, Tuo Zhang, Chenyan Lv, and Guanghua Zhao Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b01365 • Publication Date (Web): 17 May 2019 Downloaded from http://pubs.acs.org on May 18, 2019

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Designed 2D and 3D Protein Nanocage Networks Driven by Hydrophobic Interactions Contributed by Amyloidogenic Motifs Bowen Zheng, Kai Zhou, Tuo Zhang, Chenyan Lv, Guanghua Zhao* Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science & Nutritional Engineering, China Agricultural University, Beijing Key Laboratory of Functional Food from Plant Resources, Beijing 100083, China.

*Corresponding author: Guanghua Zhao, E-mail: [email protected], Phone: 0086-1062738737

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ABSTRACT: Precise manipulation of protein self-assembly by noncovalent interactions into programmed networks to mimic naturally occurring nanoarchitectures in living organisms is a challenge due to its structural heterogeneity, flexibility, and complexity. Herein, by taking advantage of both the hydrophobic forces contributed from “GLMVG” motif, a kind of amyloidogenic motifs (AMs), and the high symmetry of protein nanocages, we have built an effective protein self-assembly strategy for the construction of 2D or 3D protein nanocage arrays. According to this strategy, “GLMVG” AMs from β-amyloid 42 (Aβ42) was grafted on the outer surface of 24-meric ferritin nanocage close to its C4 symmetry channels, initially resulting in the production of 2D nanocage arrays as subgrade, and ultimately generating 3D highly ordered arrays with a simple cubic packing pattern as reaction time increases. More importantly, the reversibility and the formation rate of these protein arrays can be modulated by pH. This work provides a de novo design strategy for accurate control over 2D or 3D protein self-assemblies.

KEYWORDS: hydrophobic interactions, amyloidogenic motifs, protein nanocage surfaces redesign, pH-manipulated, 2D ordered arrays, 3D cubic-like arrays


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Proteins exhibit the capacity of self-assembly to endow themselves myriad functions during evolution. Periodic assemblies of proteins, a cornerstone of life, provide enhanced biological functions and physical–chemical properties, which contain one-dimensional (1D) microtubes, 2D bacterial surface layers and 3D virus capsid.1 Proteins represent extremely versatile building blocks for controllable self-assembly materials as their sophisticated topological structures and broad functions such as signal transmission, molecular recognition, and catalysis.2 By contrast with DNA3 and peptides4 as building blocks, designing protein selfassemblies has largely been inaccessible, plagued by the complexity of protein-protein interactions and chemical heterogeneity. To realize protein self-assembly, several strategies were adopted to implement protein self-assembly,5-6 including symmetric fusion-based selfassembly,7 utilization of metal coordination or other covalent bond,8 addition of cross-linking oligomers containing DNA or peptide,9 gene fusion approach,10 and computationally protein design.11 It has been well known that noncovalent interactions such as hydrogen bonding, electrostatic interactions, and hydrophobic interactions are mainly responsible for the formation of natural protein architectures, but, to date, constructing artificially hierarchical protein nanostructures by noncovalent interactions, especially hydrophobic interactions, has been much less as compared to those by covalent bonds (disulfide bond and metal coordination bond). To establish an effective approach for protein self-assembly driven by hydrophobic interactions, we have sought clues in the living kingdom. Nature provides numerous cases of


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protein self-assemblies, such as square arrays of aquaporin-4 (a water channel) concentrated in astrocyte endfeet,12 and gap-junction plaques in muscle and heart tissue.13 Besides the above self-assemblies occurring under normal conditions, protein assemblies also arise in diseases, such as fiber bundle of hemoglobin variant in Sickle cell anemia,14 β-amyloid (Aβ) aggregation and neurofibrillary tangles of Tau protein in Alzheimer’s disease (AD).15 These fibril formation is generally driven by amyloidogenic motifs (AMs), such as islet amyloid polypeptide fibril formation induced by “NFGAIL” motif in Type II diabetes,16 Aβ aggregation by “KLVFFAE” and “GLMVG” motifs in Alzheimer’s Disease,17 serum amyloid by “SFFSFLGEAFD” motif in Chronic inflammation amyloidosis,18 and calcitonin fibrils by “DFNKF” motif in Thyroid carcinoma.19 The AMs have been utilized to construct fibril formation of peptides or proteins,20 fabrication of nanowires21 and other molecular engineering.22 However, to date, employing AMs to construct highly ordered 2D to 3D protein nanocage arrays has yet to be explored. We are interested in AMs induced Aβ deposition which occurs at endosomal/lysosomal system, which is a relatively acidic microenvironment.23 It has been established that AMs such as “GLMVG” motifs provide the driving forces from the structural perspective at two different levels: one is the hydrophobic force between these motifs, and another is the complementary shape of grafted motifs that form adjacent ridges and grooves.24 Inspired by Aβ aggregation and the high symmetry of protein building blocks, we suppose that highly ordered 2D and 3D protein self-assembly could be constructed by controlling pH once the graft of AMs related to the formation of Aβ on the outer surface of a targeted protein is combined with the protein


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symmetry. This rational design strategy is referred as to protein surfaces redesign by the graft of AMs (GLMVG), which can be performed according to following steps: (1) Analyzing the symmetry of a scaffold protein by using crystal structural information as a guide, especially its symmetry axes such as C3, C4, or C6; (2) Determining the sites on the protein outer surface nearby these symmetry axes, which can facilitate forming complementary shape without steric clash; (3) Grafting AMs motif on the exterior surface of a targeted protein building block. Herein, we applied this strategy to implement the construction of ferritin nanocages (HFGMG) 2D to 3D arrays by the graft of “GLMVG” motifs on protein outer surface close to the

C4 symmetry axes as shown in Figure 1.

Figure 1. HF-GMG construction and its amyloidogenic motif mediated self-assembly modes. (a) Close-ups view of the four-fold symmetry axis of ferritin nanocage. The three C4 symmetry axes of ferritin nanocage are analogous to the X-Y-Z coordinate axis with the cavity center as the origin of coordinate. (b) Schematic representation of the reversible self-assembly of HF-


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GMG nanocages where the motifs “33GLMVG37” (colored by orange) from Aβ42 (colored by grey) were grafted on the exterior surface of ferritin nearby its C4 symmetry axes. The 2D and 3D networks of HF-GMG nanocages can be constructed at pH 5.7 or 6.5, which are disassociated at pH 9.0. Various protein nanocages are attractive building blocks for nanomaterials due to their chemistry and architecture diversity and inherent functions such as CO2 fixation by carboxysomes,25 nucleic acid storage and transport by viral capsids,26 and iron metabolism by ferritin.27 Because of their cage-like morphology and symmetrically structure, these protein nanocages have attracted considerable attention in the field of nanoscience and nanotechnology. Among the protein nanocages, ferritin, especially recombinant human H chain ferritin (rHuHF), exhibits high affinity to cancer cells which overexpress H-type ferritin receptor, TfR1,28 so it has emerged as a prevalent vehicle for anticancer drugs, nutrients, and tumor-imaging agents.29 Besides, rHuHF is a 24-mer homopolymer with high stability and water-solubility, which can be easily expressed in E. coli. and purified. Based on these properties, we selected rHuHF as building blocks for the construction of highly ordered protein self-assembly. Ferritins share the highly conserved architecture that composed of 24 four-α-helix subunits assembling into octahedral (432) symmetry, which form a quasi-spherical shell with a 12 nm outer diameter and a 8 nm inner diameter.30 One ferritin molecule consists of six C2 rotation axes, four C3 rotation axes and three C4 rotation axes. We focused on the C4 rotation


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axes of ferritin because they are corresponding to the space coordinates X-Y-Z when the cavity center of the quasi-spherical shell is set as the origin of the coordinates. By analyzing the crystal structure of rHuHF, we found that DE loops (159GAPES163) are located on the exterior surfaces along the C4 rotation axes (Figure 1a). Therefore, we envisioned that 2D or 3D nanocage arrays would be constructed along the C4 rotation axes after

159GAPES163

were

replaced by “GLMVG” motif from Aβ42 peptide (Figure 1b) based on the consideration that the length of this motif is approximately equal to that of the DE loops, and that the motifs form ridges and grooves that construct complementary shape to fit the conjunction between ferritin molecules.24 To confirm this idea, we made a ferritin mutant named HF-GMG where the DE loops (159GAPES163) of rHuHF were replaced with 159GLMVG163 (Figure S1), and subsequently this protein was purified to homogeneity by nearly the same method as wild-type ferritin (wt ferritin) as suggested by SDS- and Native- PAGE (Figures S2a, b). Since Aβ aggregation frequently occurs at an acidic microenvironment,21 we subsequently investigated the selfassembly of HF-GMG molecules as a function of time at an acidic pH value 5.7 by transmission electron microscopy (TEM) with a high pH value (9.0) as control. At 0.5 h, TEM analyses showed that HF-GMG nanocages are monodispersed at 9.0 (Figure S3a), while they selfassemble into 2D ordered arrays at pH 5.7 (Figure S3b). The large difference in protein assembly between pH 9.0 and 5.7 might be derived from the fact that ferritin cages take much less charges at pH 5.7 than 9.0 as suggested by zeta potential measurements (Figure S4).


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Although protein association slightly occurs with wt ferritin molecules at pH 5.7 which is close to its theoretical isoelectric point (~5.3), resulting protein aggregates are in a relatively disordered state (Figure S5a), demonstrating that the grafted AMs are directly involved in the formation of the above observed protein 2D arrays. The above observed 2D arrays are just small-scale at 0.5 h, so we wonder whether they would self-assemble into large-scale protein nanocage arrays if we prolonged reaction time. As expected, at 2.5 h, a large scale of ferritin nanocage superlattices with rectangular shape formed as a result of their neighboring components being connected by symmetrically equivalent interactions (Figures 2a-c), whereas only a small number of wt ferritin molecules form irregular aggregates under the same experimental condition (Figure S5b). On the basis of TEM characterization of HF-GMG nanocage arrays, the corresponding Fast Fourier Transform (FFT) image was performed (Figure 2d), and the real map from invert FFT as shown in Figure 2e provided an excellent view on the structure of the nanocage arrays, which reveals the unit cell parameters: α = β = 12.6 nm, λ = 89.4° (Figure 2f). The inter-row spacing, α and β, refers to the center-to-center distance between neighbor HF-GMG nanocages, and a value of 12.6 nm is well-matched with the outer diameter of ferritin (∼12 nm).


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Figure 2. Characterization of HF-GMG 2D arrays at pH 5.7 at 2.5 h. (a) Low-magnification TEM view of protein nanocage 2D arrays. (b, c) High-magnification TEM views of 2D arrays. (d) Fast Fourier Transform (FFT) image of figure 2c. (e) Real map of invert FFT from figure 2d. (f) Structural diagram based on the reconstruction of figure 2e, where α = β = 12.6 nm, λ = 89.4°. To obtain more details, the 2D arrays were further analyzed by atomic force microscopy (AFM) with an intelligent mode. As shown in Figures 3a, b, the height of arrays is about 10.4 ± 0.3 nm, which is comparable with the outer diameter of ferritin cage (~12 nm), demonstrating that the observed HF-GMG 2D arrays are of monomolecular layer. The high-magnification view of arrays showed the unit cell parameters are α = β = 11.7 nm, λ = 89.4° (Figures 3c, d), which are in good agreement with the above TEM results (Figure 2f). These results again demonstrate the importance of AMs on the outer surface of ferritin for the generation of the observed 2D nanocage arrays.


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To determine whether the above observed protein assembly also occurs in solution, dynamic light scattering (DLS) was used to examine self-assembly of HF-GMG nanocages mediated by pH at a fixed reaction time of 2.5 h. DLS analyses showed that, at pH 9.0, HFGMG nanocages showed a monodisperse distribution with hydrodynamic radius (RH) centered at ∼7.0 nm, indicative of no protein self-assembly (Figure S6a). By contrast, large species appeared in HF-GMG solution upon solution pH decreased to 5.7, although small particles with RH of ∼7.0 nm were not completely disappeared (Figure S6b), a finding confirming the above TEM and AFM results.

Figure 3. Characterization of 2D arrays by atomic force microscopy (AFM). AFM lowmagnification views of 2D HF-GMG arrays (a) and the height characterization of the 2D arrays (b). AFM high-magnification views of 2D HF-GMG arrays (c) and the corresponding height characterization along the white arrow (d).


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Interestingly, when we continued to prolong reaction time to 4 h, followed by TEM observation, we found that HF-GMG nanocage self-assemble into 3D arrays at 5.7 (Figure S7a). The self-assembly of HF-GMG nanocages at pH 5.7 was approved by DLS measurements showing that the RH of all the particles range from 354 nm to 1651 nm (Figure S7b). To obtain more insights into the above observed self-assemblies, these 3D arrays were analyzed by AFM with an intelligent mode. As shown in Figure S8, the largest height of the arrays is about 114.4 nm, suggesting that these constructed 3D structures contain up to 10 layers of ferritin nanocages. In contrast, wt ferritin molecules just slightly associated under the same conditions, resulting in the production of a small amount of aggregates in a disordered state (Figure S9a). Thus, it appears that the conversion of 2D to 3D HF-GMG protein nanocage superlattices can be simply carried out by controlling reaction time. To determine the arrangement mode and unit cell parameters, HF-GMG 3D arrays formed at pH 5.7 were analyzed by small angle X-ray scattering (SAXS). The HF-GMG sample was presented as centric diffraction rings with strong Bragg peaks in the 2D SAXS data (Figure 4a, inset image), which manifested that HF-GMG nanocages self-assemble into ordered array structures, which is achieved by genetically grafted a motif, “GLMVG”, onto protein exterior surface nearby the C4 symmetry axes. 1D SAXS data of HF-GMG arrays are dominated by intensive diffraction peaks (Figure 4a), which are well-matched with the characterization of a simple cubic structure. The identified q values (0.05213, 0.07423, 0.09037, 0.10453 Å-1) present the first four peaks ((𝒉𝒌𝒍)


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= (100), (110), (111), (200)), and the q values of sample were well coincided with q: q* =1: 2: 3 : 4. Moreover, the lattice constant 𝑎 = 11.6 nm, corresponding the center-to-center distance between the simple cubic lattice, is consistent with the exterior diameter of ferritin (∼12 nm). Based on above SAXS data, we believe that HF-GMG 3D nanocage arrays self-assemble along the C4 rotation axes into the simple cubic structure (space group P432).31 To the best of our knowledge, forming 3D ordered protein lattice driven by AMs represents the first case. HFGMG arrays with the simple cubic packing (Figure 4b) validated our design with high accuracy. Thus, the strategy by which protein outer surfaces close to the symmetry axes is grafted by AMs provides an alternative approach to construct protein nanocage arrays. Besides pH 5.7, we also investigated whether HF-GMG can self-assemble into highly ordered protein superlattices at other acidic pH values such as 6.5. We observed that HF-GMG molecules stayed in a monodispersed state initially, and then they associated with each other to form a small scale of protein assemblies after 12 h. Such small pieces of protein assemblies continually associated to generate a large scale of 2D protein arrays at 24 h; as reaction time increased to 48 h, self-assembly reaction further ultimately produced cubic-like 3D arrays (Figure 4c). As expected, just a small scale of irregular aggreates occurred with wt ferritin molecules under the same experimental conditions (Figure S9b). The formation kinetics of HFGMG 3D networks at pH 6.5 observed by TEM is in good agreement with results obtained by light scattering intensity mearsurements, namely, the self-assembly process of HF-GMG nanocages induced by pH 6.5 is much slower than that by pH 5.7 (Figure S10). This is not


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surprising because more charges on ferritin surface are screened at pH 5.7 than 6.5 as suggested by zeta potential measurements (Figure S4). Thus, it appears that HF-GMG nanocages selfassembly is very sensitive to solution pH especially at acidic microenvironment. The utilization of pH to control HF-GMG nanocage self-assembly is attractive from structural perspective at two different levels: (1) Conversion of 2D into 3D HF-GMG nanocage assembly could be controlled by changing pH; (2) The formation rate of 2D or 3D protein cage arrays is also highly pH dependent. However, it has to be mentioned that the mechanism by which 3D protein nanocage arrays are formed at pH 6.5 is similar to that at pH 5.7, that is, HF-GMG molecules firstly form 2D arrays, and then these 2D arrays interact with each other, finally resulting in the production of 3D cubic-like nanocage arrays. One overarching goal of synthetic biomimetic materials is to create hierarchical assemblies that respond to external stimuli such as pH and temperature by changing their chemical structure in response to environmental cues, so we further analyzed the response of the above protein nanocage arrays to external stimuli. We firstly investigated the reversibility of protein nannocage arrays regulated by pH. DLS analyses revealed that the above observed protein nanocage arrays formed at acidic pH environment completely disappeared when pH was increased to 9.0; instead, only monodispersed ferritin nanocages occur in solution (Figure S11a). In contrast, upon adjusting pH from 9.0 back to 5.7, such protein assembly can be produced again (Figure S11b). A similar observation was obtained for the protein assembly in solution under the same experimental condition except that pH 5.7 was replaced by 6.5


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(Figures S11c, d). These findings were approved by TEM analyses (Figure S12). Essentially, HFGMG molecules remain monodispersed at pH 9.0, which is mainly attributed to the charge-charge interactions. However, most charges on the HF-GMG surface are screened at pH 6.5 or 5.7, because such acidic pH value is close to the theoretical isoelectric point of rHuHF (~5.3), therefore the main driven force at acidic condition for triggering protein assembly is the designed hydrophobic interaction. Hence, the reversibility of HF-GMG molecule assembly can be manipulated by pH. It has been known that thermal treatmemt usually causes denaturation of most proteins due to exposure of buried nonpolar amino acid residues which, consequently, leads to protein aggregation through hydrophobic interactions, and such process is thermally irreversible. In contrast, the formation of the HF-GMG 2D and 3D netwoks driven by the hydrophobic interactions coming from AMs is reversible, which can be easily controlled by pH under mild conditions. Secondly, to investigate the thermostability of the 3D arrays at pH 6.5, they were thermaltreated at different temperatures (room temperature, 60 °C, 80 °C, and 90 °C) for 15 min, respectively, followed by analyses by TEM. Results showed that the 3D nanocage ordered arrays of HF-GMG kept well at room temperature (Figures S13a, b) and 60 °C (Figures S13c, d). In contrast, when tremperature was elevated to 80 °C, the protein nanocage 3D arrays was damged to some extent, but their cubic-like morphology was still seen (Figures S13e, f). At 90 °C, most of the cubic-like arrays disappeared; instead disordered protein assemblies occured (Figures S13g, h). Thus, it appears that HF-GMG 3D nanocage ordered arrays can endure up to 80 °C at pH 6.5. In addition, we also investigated the thermostability of the 3D arrays in


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water. TEM resutls showed that the integrated cubic-like shape of the 3D arrays can only occur at room temperature (Figures 14a, b). In contrast, these 3D nanocage arrays were almost completely collapsed, most of which were disassociated into ferritin monmers upon thermaltreated at 60 °C or 80 °C for 15 min (Figures S14c, d), indicating that the thermostability of the 3D arrays in buffer at pH 6.5 is much higher than that in water.

Figure 4. The pattern of HF-GMG 3D arrays formation. (a) SAXS analyses of HF-GMG 3D arrays. The (hkl) values in radially averaged 1D SAXS data are shown above the peaks. Experimental curve (colored by black) is well-matched with simulated pattern (shown in red), which indicates that HF-GMG 3D array is a simple cubic structure according to the equation 𝑞 =

2𝜋 𝑎

ℎ2 + 𝑘2 + 𝑙2. The inset image is the 2D SAXS pattern of HF-GMG 3D self-assemblies. (b)

Structure model of the simple cubic architecture corresponding to (a). (c) Low- and Highmagnification TEM views of HF-GMG self-assemblies as a function of time at pH 6.5.


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A major hurdle to be overcome in exerting accurate control over protein self-assemblies is that protein-protein interactions are mediated by weak, noncovalent interactions over large surfaces. We recently built 1D, 2D and 3D protein nanocage arrays by using electrostatic interactions32-33 and π-π stacking interactions,34 respectively, but in this work, inspired by Aβ aggregation, we utilized hydrophobic interctions as a driving force in a combination with the protein symmetry to construct protein nanocage arrays, which represents an effective, easy solution to control over protein self-assemblies. Based on our strategy, highly ordered 2D and 3D protein nanocage arrays can be generated through a bottom-up strategy in a predictable way. More importantly, the formation of such highly ordered 2D and 3D ferritin nanocage arrays as well as the conversion between these two different arrays can be regualted easily by changing eithor pH in solution or reaction time. Besides the “GLMVG” motif from Aβ42 peptide, other naturally occurring AMs under normal or disease-realted conditions, can also be grafted onto the exterior surface of scaffold proteins. Therefore, our protein engineering strategy can, in principle, be applied to other protein building blocks with the high symmetry (including C3 or higher symmetry axes) such as Dps,35 viral capsid,36 and other artificial protein nanocages37 or nanorings,38 resulting in the generation of a variety of highly ordered self-assemblies with different properties.


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ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. The SI includes details of the experimental methods and amino acid sequence of wt ferritin and HF-GMG, SDS- and Native- PAGE of wt ferritin and HF-GMG, TEM views of wt ferritin as control group at the same experimental environment, zeta potential measurements at different pH, DLS measurements of HF-GMG 2D and 3D arrays solution at different pH and different reaction time, and reversibility and the formation rate of 2D or 3D HF-GMG arrays at different pH. (PDF) Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by National Key R&D Program of China (2018YFD0901004) and the National Natural Science Foundation of China (No. 31730069). We also thank the staffs in Tsinghua University Branch of China National Center for Protein Sciences Beijing for technical assist, and the staffs in Institute of Chemistry Chinese Academy of Sciences. AUTHOR INFORMATION Corresponding Author Guanghua Zhao*


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E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. All these authors contributed to this work. Guanghua Zhao supervised the research. Guanghua Zhao and Bowen Zheng conceived and designed the experiments. Bowen Zheng, Kai Zhou, Tuo Zhang and Chenyan Lv prepared the samples and performed the experiments. All authors wrote the paper, discussed the results, and commented on the manuscript. Funding Sources National Key R&D Program of China (2018YFD0901004) and National Natural Science Foundation of China (No. 31730069). ABBREVIATIONS AMs, amyloidogenic motifs; Aβ42, β-amyloid 42; rHuHF, recombinant human H-type ferritin; wt ferritin, wide-type ferritin (it refers to rHuHF in this work); GMG motifs, “GLMVG” motifs; HF-GMG, rHuHF variant with GMG motifs; TEM, transmission electron microscopy; DLS, dynamic light scattering; AFM, atomic force microscope; SAXS, small angle X-ray scattering.


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TOC graphic Designed 2D and 3D Protein Nanocage Networks Driven by Hydrophobic Interactions Contributed by Amyloidogenic Motifs

TOC 1. 3D protein nanocage arrays construction and its amyloidogenic motif mediated selfassembly modes. Herein, by taking advantage of both the hydrophobic forces contributed from the “GLMVG” motifs, a kind of amyloidogenic motifs (AMs), and the high symmetry of protein nanocages, we have built an effective protein self-assembly strategy for the construction of 2D or 3D protein nanocage arrays. The reversibility and the formation rate of these protein arrays can be modulated by pH.


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Designed Two- and Three-Dimensional Protein Nanocage Networks

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