Self-Assembled Materials Made from Functional Recombinant

Sep 28, 2016 - Porphyromonas gingivalis gingipains: The molecular teeth of a microbial vampire. O'Brien-simpson, N. M.; Veith, P. D.; Dashper, S. G.; ...
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Self-Assembled Materials Made from Functional Recombinant Proteins Yeongseon Jang and Julie A. Champion* School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, 950 Atlantic Drive NW, Atlanta, Georgia 30332, United States CONSPECTUS: Proteins are potent molecules that can be used as therapeutics, sensors, and biocatalysts with many advantages over small-molecule counterparts due to the specificity of their activity based on their amino acid sequence and folded three-dimensional structure. However, they also have significant limitations in their stability, localization, and recovery when used in soluble form. These opportunities and challenges have motivated the creation of materials from such functional proteins in order to protect and present them in a way that enhances their function. We have designed functional recombinant fusion proteins capable of self-assembling into materials with unique structures that maintain or improve the functionality of the protein. Fusion of either a functional protein or an assembly domain to a leucine zipper domain makes the materials design strategy modular, based on the high affinity between leucine zippers. The self-assembly domains, including elastin-like polypeptides (ELPs) and defined-sequence random coil polypeptides, can be fused with a leucine zipper motif in order to promote assembly of the fusion proteins into larger structures upon specific stimuli such as temperature and ionic strength. Fusion of other functional domains with the counterpart leucine zipper motif endows the self-assembled materials with protein-specific functions such as fluorescence or catalytic activity. In this Account, we describe several examples of materials assembled from functional fusion proteins as well as the structural characterization, functionality, and understanding of the assembly mechanism. The first example is zipper fusion proteins containing ELPs that assemble into particles when introduced to a model extracellular matrix and subsequently disassemble over time to release the functional protein for drug delivery applications. Under different conditions, the same fusion proteins can selfassemble into hollow vesicles. The vesicles display a functional protein on the surface and can also carry protein, small-molecule, or nanoparticle cargo in the vesicle lumen. To create a material with a more complex hierarchical structure, we combined calcium phosphate with zipper fusion proteins containing random coil polypeptides to produce hybrid protein−inorganic supraparticles with high surface area and porous structure. The use of a functional enzyme created supraparticles with the ability to degrade inflammatory cytokines. Our characterization of these protein materials revealed that the molecular interactions are complex because of the large size of the protein building blocks, their folded structures, and the number of potential interactions including hydrophobic interactions, electrostatic interactions, van der Waals forces, and specific affinity-based interactions. It is difficult or even impossible to predict the structures a priori. However, once the basic assembly principles are understood, there is opportunity to tune the material properties, such as size, through control of the self-assembly conditions. Our future efforts on the fundamental side will focus on identifying the phase space of self-assembly of these fusion proteins and additional experimental levers with which to control and tune the resulting materials. On the application side, we are investigating an array of different functional proteins to expand the use of these structures in both therapeutic protein delivery and biocatalysis.

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INTRODUCTION Self-assembly is a process in which components of a system spontaneously assemble into an ordered supramolecular structure through non-covalent intermolecular interactions such as hydrogen-bonding, van der Waals, Coulombic, or hydrophobic interactions.1 Self-assembly has been widely used in materials science, chemistry, and biology as a bottom-up approach to create ordered structures at the nanometer to micrometer scale with high complexity. Many researchers have been particularly inspired by natural structures such as cells, shells, and the extracellular matrix, which result from self-assembly of basic biological building blocks including nucleic acids, lipids, and proteins. A diverse © 2016 American Chemical Society

array of self-assembled architectures have been made from these functional biomolecules to fabricate novel supramolecular structures not found in nature.2 For example, DNA has been used as a nanobuilding block for self-assembled materials3 based on the exquisite specificity of Watson−Crick base pairing, which allows the design of a large set of nucleotide sequences with predictable binding interactions. DNA origami methods have been developed with these sequences to generate a wide variety of ordered structures in many different dimensions.4,5 Peptides and proteins that are composed of one or more long Received: June 30, 2016 Published: September 28, 2016 2188

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Figure 1. Recombinant fusion proteins containing leucine zipper motifs. (A) Rigid heterodimers made from acidic ZE and basic ZR via strong interactions with femtomolar affinity. (B, C) Recombinant fusion proteins containing (B) ZR (ZR−ELP and ZR−C10−ZR) and (C) ZE (mCherry−ZE, EGFP−ZE, and pRgpACAT−ZE) described in this Account. (D) Schematic illustration of the protein expression and purification process.

globular proteins to create more complex self-assembled structures containing the functionally folded proteins is of significant interest but is still in progress because globulardomain-containing fusion proteins have different thermodynamics in self-assembly compared with free globular proteins because of their folded shapes and specific interactions.14−16 Variations in amino acid sequence between the domains in a fusion protein can confer a high degree of specificity in the intramolecular interactions and its molecular structure,17 whereas the collection of a variety of such domains into multidomain polypeptides enables the generation of assembled structures with greater complexity. New sequences continue to be identified, and more elaborate molecular design might permit more complicated biomolecular assemblies.6,18 In addition, considerable efforts have recently been made to map the phase behavior of diverse types of recombinant fusion proteins to create self-assembled supramolecular materials with tunable structures and sizes.14,19 Despite these examples, devising chemical strategies to create well-controlled structures from protein self-assemblies is still in the early stages because of the greater complexity of protein− protein interactions. Therefore, more work is necessary to understand the physical and chemical mechanisms that govern the assembly and function of protein structures in order to develop innovative protein-based materials from specifically designed proteins. In this regard, this Account will focus on the role of recombinant fusion protein building block units in their self-assembly based on detailed characterization of the structures and properties of the self-assembled materials made from the fusion proteins. The recombinant fusion proteins

chains of amino acid sequences have also served as basic building blocks for biofunctional supramolecular structures. Proteins provide variability in self-assembled structures based on the formation of secondary, tertiary, and quaternary structures as well as specific affinity binding interactions. Fibers, tubes, rings, cages, and more complex structures have been constructed by the self-assembly of diverse peptides and proteins with unique primary sequences.6 In addition, proteins can provide biological functions in self-assembled structures, such as biocatalysis and response to stimuli, based on specifically folded three-dimensional structures that dictate protein activity.7−9 The biggest advantage of using proteins as self-assembling building blocks is that the protein domains or peptide sequences can be designed to confer specific functions to engineered biomaterials via recombinant technology.10 The desired recombinant proteins are designed at the DNA level and translated with high fidelity so that different protein domains can be assembled in the desired order with a specified number of repeats. Previous studies have shown the possibility of creating self-assembled materials with new functionalities by combining two structural protein elements via recombinant protein engineering. For example, a new biomaterial to mimic the mechanical properties of muscles was designed through the combination of the well-characterized protein domains GB112 and resilin.11 Also, many studies of recombinant fusion proteins containing elastin-like polypeptide (ELP) sequences with thermally induced inverse phase transition behavior have reported a range of temperature-responsive self-assembled materials for various biomedical applications.12,13 Fusion of 2189

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proteins serve as hydrophilic blocks connected to the rodshaped leucine zippers and can be used as models for functional proteins in self-assembly of protein materials. We also extended the set of model proteins to include a bioactive enzyme, RgpA, to create pRgpACAT−ZE. RgpA is an arginine-specific gingipain produced by Porphyromonas gingivalis that degrades inflammatory cytokine tumor necrosis factor α (TNF-α) and therefore has therapeutic potential.28

described in this Account contain coiled-coil leucine zippers. These serve as the link between functionally folded globular proteins, such as fluorescent proteins or enzymes, and building block domains with self-assembly properties.

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BUILDING BLOCKS FOR SELF-ASSEMBLED PROTEIN MATERIALS One challenge with creating self-assembled materials with functional proteins is that the identity and properties of the functional protein can significantly affect the final material structure and properties. This could require re-engineering for each new functional protein. Therefore, we sought to utilize a modular approach that would allow for more general materials design. This was achieved through the use of a rigid heterodimer leucine zipper pair made from the glutamic acidrich, acidic motif ZE and the arginine-rich, basic motif ZR. ZE and ZR have exceptionally high affinity (dissociation constant Kd ≈ 10−15 M),20 and the pair forms stable and precise complexes under physiological conditions.21 With these, we designed recombinant fusion proteins consisting of a leucine zipper motif (ZR or ZE) and either a functional protein or an “assembly” domain in order to construct various self-assembled structures based on the high affinity between the zippers (Figure 1A). The assembly domains used in this work are an ELP and an artificial random coil block (C10), which were conjugated with ZR domains (Figure 1B). The ZR−ELP and ZR−C10−ZR proteins served as “assembling protein building blocks” in self-assembled materials. We fused the ZE domain with the model fluorescent proteins mCherry and enhanced green fluorescent protein (EGFP) as functional proteins, and recent work has expanded the toolbox to include bioactive enzymes such as RgpA (Figure 1C). We used the functional fusion protein building blocks (mCherry−ZE , EGFP−ZE, and pRgpACAT−ZE) in order to characterize and functionalize the resulting self-assembled materials via the leucine zipper domains. The fusion proteins were produced in Escherichia coli and purified by affinity chromatography (Figure 1D). Each domain in our protein building blocks is described below. ELPs are derived from tropoelastin and contain repeats of five amino acids, Val-Pro-Gly-Xaa-Gly (VPGXG), where X can be any amino acid except proline.22 They exhibit lower critical solution temperature (LCST) behavior and undergo an inverse phase transition from soluble to insoluble in aqueous solution above a certain transition temperature.22−24 Therefore, they can serve as hydrophobic building block units during self-assembly of fusion protein complexes above the transition temperature. When ELPs are combined with hydrophilic domains, the resulting amphiphilic ELP fusion proteins can form diverse types of self-assembled structures such as spherical micelles,24 wormlike micelles,25 and vesicles.26 We designed an amphiphilic fusion protein, ZR−ELP, by combining the pentarepeat ELP [(VPGVG)2VPGFG(VPGVG)2]5 with ZR.15,16 C10 is an artificial random coil block of an alanylglycine (AG)-rich repeat combined with proline and glutamic acid residues, [(AG)3PEG]10.27 While it does not have any inherent assembly properties, we hypothesize that the oxygen atoms of carboxylic groups in the glutamic acid residues and its flexible backbone can allow coordination with calcium ions (Ca2+). We designed a triblock fusion protein that contains two ZR motifs with C10, ZR−C10−ZR. For functional domains, we employed globular fluorescent proteins to make mCherry−ZE and EGFP−ZE. The fluorescent

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IN SITU ASSEMBLY OF ZR−ELP AND MCHERRY−ZE PARTICLES An in situ self-assembly system of ZR−ELP and mCherry−ZE was developed in which the engineered fusion proteins selfassemble when introduced into a model extracellular matrix (ECM).16 The two-component system made from selfassembly based on the leucine zippers can provide local delivery of protein therapeutics to diseased sites to maintain local concentration over a prolonged period of time. Soluble ZR−ELP building blocks self-assemble into coacervated particles upon heating to body temperature as a result of the temperature-induced inverse phase transition of the ELP domain. Fluorescent mCherry−ZE proteins serve as model proteins to be delivered to and dissociated from the ZR−ELP assembled particles in an ECM. With these, we investigated the in situ assembly of ZR−ELP and mCherry−ZE in a model ECM and found it to show different characteristics in the molecular transport of the fusion proteins compared with aqueous solution because of their limited diffusivity in the constrained matrix. Before self-assembly in the ECM was accomplished, the assembly properties in solution were established. The inverse phase transition of ZR−ELP upon temperature elevation was characterized by an increase in turbidity in aqueous solution (Figure 2A).16 The transition temperature of ELP fusion proteins is dependent on intrinsic factors such as the length and sequence of the ELP and the polarity of other blocks fused with the ELP29 as well as external factors such as the ionic strength and ELP concentration.15,16,30 ZR−ELP in the concentration range of 1.9 to 2.4 μM in phosphate-buffered saline (PBS) containing 137 mM NaCl formed coacervates when the solution was heated from 25 to 37 °C, and the rapid binding of mCherry−ZE to the ZR−ELP coacervates was observed within a minute. In an aqueous solution, ZR−ELP grows into microspheres with smooth surfaces due to the free coacervation−coalescence process, as has been reported for other ELPs.25,31,32 The particle morphology is not altered by the binding of mCherry−ZE (Figure 2B,C).16 We accomplished the two-step self-assembly of the fusion protein building blocks (ZR−ELP and mCherry−ZE) in a reconstituted ECM hydrogel (Matrigel), which is used as a model for native ECM (Figure 3A).16 First, soluble ZR−ELP below the transition temperature (25 °C) permeated through the ECM hydrogel (37 °C) and formed coacervated protein particles in situ. In the second step, mCherry−ZE protein was added into the ECM to induce its binding to the ZR−ELP coacervates. The mCherry−ZE/ZR−ELP particulates selfassembled via strong ZE/ZR affinity and showed irregular shapes in the ECM with diameters ranging from 330 nm to 1.6 μm (Figure 3B,C), indicating the incomplete coalescence of the coacervated ZR−ELP nanoparticles in the hydrogel matrix.16 The ECM impeded the diffusion of ZR−ELP coacervates, preventing the coalescence and growth of large spherical particles as seen in solution.31 The characteristic that ELP 2190

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molecules for cancer therapy.33 The diffusivity of mCherry−ZE and mCherry−ZE/ZR−ELP particles in Matrigel or PBS was determined by obtaining fluorescence intensity profiles as a function of time and fitting the data to a Fickian diffusion model. As a result, mCherry−ZE/ZR−ELP particles had 2.5-fold lower diffusivity in ECM hydrogel than in PBS. Also, the assembled particles presented 500-fold lower diffusivity compared with free mCherry−ZE in the hydrogel (Figure 3D).16 In this regard, the in situ self-assembly can result in entrapment of a functionally folded protein such as mCherry in ECM hydrogel by the self-assembly process. Furthermore, the self-assembled protein particles trapped in the ECM dissociate and are released at a controlled rate. The insignificant electrostatic interactions of the fusion proteins with the ECM enabled mCherry−ZE/ZR−ELP particles to be locally dynamic with reduced mobility but not immobilized on the matrix. The self-assembled particles dissociated over time in ECM hydrogel, and we hypothesized that this occurred because diffusion led the local concentration of ZR−ELP to drop below the minimum concentration for the phase transition (1.9 μM at 37 °C). Consequently, the average diameter as well as the polydispersity of the particulates decreased over time (Figure 4). The dissociation of ZR−ELP from the self-assembled particles also induced an initial release of mCherry−ZE (36% loss over 12 h), but it was much slower than that of soluble mCherry−ZE (58% loss).16 The self-assembled particles of mCherry−ZE/ZR−ELP formed in the ECM presented different shape, size, and dissolution kinetics compared with those in an aqueous solution. The proteins could be topically applied or injected to the target tissues in the soluble phase and the in situ assembly process starts with contact at warm body temperature. This system could be useful as carrier-free delivery system of engineered biocompatible proteins via self-assembly and selfdissociation induced by the temporal concentration gradient in the ECM.

Figure 2. (A) Inverse phase transition of ZR−ELP in aqueous solution. The optical density (OD) at 350 nm was measured at different concentrations of ZR−ELP (1, 2, 4, 8, 16, and 32 μM) as a function of temperature (heating rate = 1 °C/min). (B) Fluorescence micrograph of mCherry−ZE/ZR−ELP particles assembled in aqueous solution. (C) Changes in the fluorescence intensity profiles of the particles within 1 min of incubation. Reprinted with permission from ref 16. Copyright 2013 Wiley-VCH GmbH & Co. KGaA, Weinheim.

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VESICLE FORMATION FROM ZR−ELP AND GLOBULE−ZE Vesicles, defined here as enclosed compartments filled with a small amount of fluid, have advantages as drug delivery vehicles for medical applications. They can carry multiple cargoes such

forms viscous, gel-like coacervates upon injection to tissues can be utilized to develop biodegradable and injectable localized protein drug delivery systems, similar to those delivering small

Figure 3. (A) Schematic representation of the in situ assembly and disassembly of ZR−ELP and mCherry−ZE fusion proteins in an extracellular matrix (ECM). (B) Fluorescence micrographs of self-assembled mCherry−ZE/ZR−ELP in model ECM (Matrigel) (inset scale bar: 1 μm). (C) Scanning electron micrographs of protein particles embedded in Matrigel (inset scale bars: 5 μm). (D) Diffusivity of soluble mCherry−ZE (mChZ) or self-assembled particles (SAPs). Reprinted with permission from ref 16. Copyright 2013 Wiley-VCH GmbH & Co. KGaA, Weinheim. 2191

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components to achieve the correct molecular packing. Although ELP fusion proteins have been reported to form solid coacervate particles, micelles or vesicles from self-assembly or template processes,25,26,31,32,34 there is no report of globular proteins incorporated in self-assembled vesicles. We reported template-free vesicle formation from thermally induced self-assembly of ZR−ELP and globule−ZE (i.e., mCherry−ZE and EGFP−ZE) above critical values of the salt concentration.15 The self-assembled structures from ZR−ELP and globule−ZE can be controlled by not only the temperature but also the molar ratio of globule−ZE to ZR−ELP and the salt concentration in aqueous buffers. In addition, the self-assembly characteristic of the protein building blocks that depends on the salt concentration enables the formation of core−shell structures showing separation of two globular proteins into different phases within a vesicle. The globule−ZE proteins formed soluble “globule−rod−coil” protein complexes with ZR−ELP at 4 °C via the high-affinity interaction of leucine zipper pairs, and ZR−ELP formed “rod− coil” protein homodimers through dimerization of ZR motifs (Kd ≈ 10−7 M) (Figure 5A).15,16 The globule−rod−coil complexes and rod−coil amphiphiles can self-assemble into coacervate particles or vesicles at 25 °C as a function of salt concentration. The mCherry−ZE/ZR−ELP mixture selfassembled into vesicles above a salt concentration of 0.3 M (Figure 5B), while EGFP−ZE/ZR−ELP vesicles were observed only above a salt concentration of 0.91 M (Figure 5C). Below the critical salt concentrations, only coacervated particles of the protein complexes were observed. Therefore, we were able to construct diverse vesicle structures incorporating multiple types of globular domains in either the membrane or the internal cavity by tuning the salt concentration. Both globular proteins (mCherry−ZE and EGFP−ZE) were located in each vesicle membrane at a salt concentration of 0.91 M (Figure 5D), whereas EGFP−ZE/ZR−ELP coacervated particles were separated into the interior compartment of mCherry−ZE/ZR−ELP vesicles at a salt concentration of 0.45 M (Figure 5E).15 The ability of amphiphilic molecules to assemble into vesicle structures is based on their shape, as described by the molecular packing parameter P = V/(a0lc),35 where V and lc are volume

Figure 4. Dissociation profiles of mCherry−ZE/ZR−ELP particles selfassembled in an ECM. (A) Changes in particle size distribution as a function of time, (B) changes in fluorescence intensity induced by mCherry−ZE dissociation, and (C) release profiles of mCherry−ZE in the ECM, as self-assembled particles (red, SAPs) or in soluble form (black, mCherry−ZE), and mCherry without the ZE motif in the presence of ZR−ELP (blue, mCherry/ZR−ELP). Adapted with permission from ref 16. Copyright 2013 Wiley-VCH GmbH & Co. KGaA, Weinheim.

as targeting ligands or therapeutic agents while providing stability of structure and functionality for biomolecules. However, direct incorporation of folded and biologically relevant proteins into vesicles is underdeveloped because of the limitation of their conformational arrangement and the need to use organic solvents.19 The difficulties of forming vesicles containing folded proteins can be overcome through the use of recombinant fusion proteins containing leucine zipper motifs and ELPs.15 The aqueous hydrophobic transition of ELP eliminated the need for the organic solvent, and the non-covalent linkage of the folded globular proteins to ELPs via the zippers enabled independent tuning of these two

Figure 5. (A) Schematic illustration of the recombinant protein amphiphiles as building blocks for self-assembly into vesicles. (B−E) Confocal micrographs of self-assembled protein vesicles of (B) mCherry−ZE/ZR−ELP at a salt concentration of 0.3 M, (C) EGFP−ZE/ZR−ELP at a salt concentration of 0.91 M, and mCherry−ZE and EGFP−ZE mixtures with ZR−ELP at different salt concentrations ((D) 0.91 M, (E) 0.45 M) (inset scale bars: 1 μm). The micrographs are reprinted from ref 15. Copyright 2014 American Chemical Society. 2192

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Accounts of Chemical Research and length of the hydrophobic ELP block, respectively, and a0 is the cross sectional area of the hydrophilic domain (Figure 6A). Since the hydrophobic folding of ELP motifs and their

result of the reduced curvature caused by increased attraction between EGFP domains (Figure 6B).15 Preferential encapsulation of EGFP−ZE/ZR−ELP coacervate in mCherry−ZE/ZR−ELP vesicles at a salt concentration of 0.45 M indicates that other hydrophobic cargoes can also be simply encapsulated into vesicles by mixing with the protein amphiphiles prior to thermal incubation above the transition temperature. As a proof of concept, we demonstrated that subnanometer-scale fluorescein and submicrometer-scale polystyrene beads can be spontaneously encapsulated into mCherry−ZE/ZR−ELP vesicles by mixing followed by the inverse phase transition (Figure 7).15 This result could be

Figure 7. Confocal micrographs of self-assembled protein vesicles encapsulating different types of cargo: (A) fluorescein and (B) FITCconjugated polystyrene particles (diameter ∼500 nm) (inset scale bars: 1 μm). Reprinted from ref 15. Copyright 2014 American Chemical Society.

explained by a hypothetical model for the vesicle structure that has a self-assembled single-layer membrane of “globule−rod− coil” complexes providing a hydrophobic inner wall. Similar to the single-layer suprastructures made from self-assembly of metal−polymer amphiphiles,38 we hypothesized that the rigid, rod-shaped conformations of ZE/ZR domains can maintain the low interfacial curvature of membranes and may prevent collapse of the hollow structure above the critical salt concentration. However, the exact structure of protein vesicle membranes warrants more detailed investigation in future work. The simple and efficient encapsulation of various types of cargoes with multiple length scales from ∼1 to 500 nm can provide potential opportunities in drug delivery applications. Furthermore, the aqueous self-assembly of protein vesicles containing globular domains as building blocks suggests a versatile method to fabricate multifunctional vesicles with diverse types of folded and biologically functional proteins such as drugs, enzymes, and targeting ligands in biocompatible environments.

Figure 6. (A) Truncated cone models of a ZR−ELP homodimer and an mCherry−ZE/ZR−ELP complex to explain the packing parameter (P) for self-assembly. (B) Hydrodynamic diameter (dH) obtained from dynamic light scattering measurements as a function of the molar ratio (χ) of globule−ZE to ZR−ELP. Reprinted from ref 15. Copyright 2014 American Chemical Society.

hydrophobic interactions are strongly affected by the ionic strength of the solution,25 the packing parameter favored for vesicle formation (1/2 < P < 1) can be achieved above the critical salt concentration. In addition, the packing parameter can explain why the critical salt concentration varies in different globular fusion proteins. The value of a0, defined as the average head area of the hydrophilic block, will be affected by the head areas of ZR/ZR (a1) and globule−ZE/ZR (a2) as well as the molar ratio of globule−ZE to ZR−ELP (χ): a0 = (1 − χ)a1/2 + χa2 (Figure 6A). The value of a2 is dictated by the nature of the globular domains fused to ZE/ZR coiled coils. In this respect, monomeric, highly soluble, charged mCherry36 can provide a larger head area (a2) than EGFP, which tends to dimerize37 and aggregate with other EGFPs. Therefore, the required salt concentration for self-assembly into a vesicle is higher in the case of EGFP fusion proteins than mCherry ones. Furthermore, the natural properties of globular fusion proteins lead to changes in the vesicle curvature as a function of the molar ratio χ. With an increase in χ, the hydrodynamic diameter of vesicles containing mCherry−ZE decreases because of the increased curvature induced by repulsive forces between mCherry domains, while the size of EGFP−ZE vesicles increases as a

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HIERARCHICAL SUPRAPARTICLES FROM ZR−C10−ZR The self-assembly of recombinant proteins containing leucine zipper motifs can be extended further to create functional hierarchical architectures with large surface areas that can be utilized for protein immobilization via the specific interaction of ZE/ZR pairs. We envision this to be ideal for enzyme immobilization. We fabricated hierarchically structured porous supraparticles from self-assembly of flowerlike nanobuilding blocks composed of the recombinant fusion protein ZR−C10− ZR and inorganic calcium phosphate (CaP).39 Structures like these are not possible from pure protein assembly, thus 2193

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Figure 8. Schematic illustrations and SEM images of (A) the formation of hybrid flower-shaped (FS) nanoparticles from ZR−C10−ZR fusion proteins and CaP and (B) the self-assembly of hybrid FS nanoparticles into chainlike clusters. Reprinted from ref 39. Copyright 2016 American Chemical Society.

requiring the hard inorganic component to assemble with proteins. The self-assembled porous supraparticles were further modified by immobilization of the pathogen-derived protease (pRgpACAT−ZE), which showed improved inactivation of tumor necrosis factor α (TNF-α) over soluble protease.28 The hierarchical structured porous supraparticles were fabricated by self-assembly of protein−inorganic hybrid building blocks. The fusion protein ZR−C10−ZR and CaP spontaneously made nanoscale flower-shaped (FS) building blocks in aqueous solution. Addition of calcium chloride (CaCl2) to a ZR−C10−ZR solution in PBS caused immediate precipitation of CaP, which is attributed to fast crystal growth, showing platelike petal structures (Figure 8A).39 We proposed that the artificial random coil block (C10) in the ZR−C10−ZR fusion protein would dominantly interact with the calcium ions by providing oxygen atoms of carboxyl groups in glutamic acid residues and a flexible backbone. On the other hand, the ZR motifs present rigid structures containing few glutamic acid residues, and would remain available for specific immobilization of ZE-containing fusion proteins within the hybrid materials. The hybrid FS nanoparticles then instantaneously selfassembled into chainlike clusters, which is thought to be mediated by attractive interactions between the FS particles and steric hindrance along the sides of cluster chains (Figure 8B).39 We hypothesized that the attractive interactions were induced by van der Waals attractions and hydrophobic interactions of the CaP/ZR−C10−ZR hybrid particles and that their multiple edges or “flower petals” could provide high-valency interactions for further assembly into clusters. The self-assembly of FS nanoparticles composed of fusion proteins into higher-order hierarchical structures is unique and beneficial for biological applications. FS particles assembled from diverse inorganic nanostructures such as CdSe nanoparticles40 or Pt nanowires41 have been actively developed.42,43 However, to our knowledge, there are no reports to date of hierarchical suprastructures self-assembled from protein-based FS structures. Furthermore, this hierarchical supraparticle formation under biocompatible conditions facilitates robust immobilization of biofunctional proteins such as enzymes compared with other methods to produce CaP supraparticles, including hydrothermal synthesis44 or the sol−gel process.45

Using the FS nanoparticle clusters, we developed a simple strategy to fabricate protein−inorganic hybrid hierarchical supraparticles from the combination of solution-phase and interfacial assembly of FS nanoparticle clusters (Figure 9A). With end-over-end rotation that provided continuous ex-

Figure 9. (A) Schematic illustration of colloidal assembly of FS nanoparticle clusters into hierarchically structured porous supraparticles. The proposed mechanism of assembly is that clusters concentrate and organize at the air−water interface and attach to the surface of seed particles as the interface is compressed. (B) Optical micrographs and (C) size distribution of supraparticles in solution. (D−F) Scanning electron microscopy (SEM) images of dried supraparticles at different magnifications (close-up SEM images of porous structures were obtained in the marked area). The optical micrographs and scheme are reprinted from ref 39. Copyright 2016 American Chemical Society. The SEM images are reprinted with permission from ref 28. Copyright 2016 The Royal Society of Chemistry. 2194

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utilized for immobilization of proteins of interest via highly specific protein−protein interactions. To further demonstrate biofunctionality, we immobilized a pathogenic cysteine protease within the supraparticles to realize functional self-assembled materials that can inactivate the inflammatory molecule TNF-α.46 Arginine-specific gingipains (Rgps) secreted from P. gingivalis prevent inflammatory responses by inactivating TNF-α through enzymatic degradation. We engineered the fusion protein pRgpACAT−ZE to be specifically immobilized within the hybrid supraparticles containing the leucine zipper motif ZR.28 Adhesion domains were removed from RgpA to reduce immunogenicity and hemagglutination of the wild-type RgpA, so that only the propeptide and catalytic domains (pRgpACAT) were fused with the leucine zipper motif ZE. The propeptide domains are required for proper folding and were autolytically cleaved from the catalytic domain after expression in E. coli (Figure 11A).28 The proteolytic activity of pRgpACAT−ZE toward FITCconjugated TNF-α was directly confirmed by a fluorescence quenching assay (Figure 11B).28 The hierarchically structured self-assembled supraparticles with pores ranging from tens of nanometers to several micrometers facilitate immobilization of functional enzymes

pansion and compression of the air−water interface of the FS nanoparticle cluster solution for 2 days, hierarchical porous supraparticles with an average diameter of 30 ± 10 μm were assembled (Figure 9B).39 Since the FS nanoparticle clusters did not assemble into supraparticles in the absence of an interface or with a constant-area interface, it was demonstrated that the assembly of FS clusters into supraparticles is mediated by interfacial adsorption and compression at the air−water interface. Petals in each FS cluster were interconnected within the assembled structures, providing nanometer-scale pores, and the self-assembly of the clusters resulted in the formation of micrometer-scale pores. As a result, the supraparticles showed hierarchical pores (Figure 9D−F). An important feature of the protein−inorganic hybrid hierarchical supraparticles is that they can provide a large surface area of specific affinity binding sites, which facilitates immobilization of functional proteins. It was confirmed that model proteins mCherry−ZE and EGFP−ZE28,39 can be selectively immobilized within the ZR-motif-containing hybrid supraparticles (Figure 10A) whereas fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) cannot be adsorbed nonspecifically (Figure 10B). Compared with CaP nanoparticles without ZR−C10−ZR, which did show nonspecific adsorption, it was demonstrated that the supraparticles can be

Figure 10. Immobilization of globule−ZE proteins within hybrid supraparticles with high specificity. (A) Center confocal slice of a supraparticle incubated with mCherry−ZE (left) and close-up images of the core (blue frame) and boundary regions (green frame) showing a spatial difference in supraparticle density (left). Immobilization of EGFP−ZE (green) within a supraparticle is also confirmed by fluorescence microscopy (right). (B) Fluorescence microscopy images of the hybrid supraparticles (top) and CaP nanoplates (bottom) incubated with both mCherry−ZE (red) and FITC−BSA (green). Reprinted with permission from refs 28 and 39. Copyright 2016 The Royal Society of Chemistry and American Chemical Society, respectively.

Figure 11. Characterization of the proteolytically active fusion protein pRgpACAT−ZE. (A) SDS-PAGE gel image of cleared lysate (CL), flowthrough (FL), washes (W1, W2), and elution (E) samples during purification of pRgpACAT−ZE. Arrows indicate (i) full length, (ii) partially cleaved, and (iii) fully cleaved pRgpACAT−ZE. (B) Proteolytic degradation of TNF-α by soluble pRgpACAT−ZE is characterized by the change in fluorescence intensity (ΔRFU) of FITC-conjugated TNF-α after incubation for 30 min. Reprinted with permission from ref 28. Copyright 2016 The Royal Society of Chemistry. 2195

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particles to hollow vesicles to hierarchical supraparticles. Rational protein design is used to introduce sequences such as ELPs or C10 that provide self-assembly capability, such as thermally induced phase transition or oxygen-rich binding sites. All of the designs incorporate leucine zippers to enable modularity to extend the material design to a larger number of functional proteins. We have performed detailed materials characterization and investigated the assembly mechanisms in order to be able to control the physical properties of the materials and extend them to new functional proteins. We are currently working to map the phase space of these protein assemblies and apply advanced tools more commonly used in polymeric materials to better understand and evolve our materials. As advances are made in computational tools initially utilized for protein folding, it is likely that they can make significant contributions to the design, prediction, and understanding of protein materials made from natural, engineered, and de novo proteins.48 Given the specific activities possible from proteins, we envision that self-assembled materials made from functional proteins could have wide applicability ranging from drug delivery and tissue engineering to biocatalysis and sensing. We are working with a wide range of functional proteins to expand the utility of the described materials and enable the design of new structures.

within the particles and enable substrate and product transport (Figure 9B−F).28,39 We successfully immobilized the bioactive pRgpACAT−ZE within the supraparticles using the specific heterodimerization of the leucine zipper pairs. The activity of catalytic domains within supraparticles was investigated by monitoring viability of a TNF-α-sensitive cell line (L929 cells) exposed to TNF-α solutions containing either soluble pRgpACAT−ZE or the pRgpACAT−ZE-immobilized supraparticles (Figure 12). Supraparticles incorporating pRgpACAT−ZE

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Figure 12. Inactivation of TNF-α by soluble RgpACAT−ZE or RgpACAT−ZE immobilized in supraparticles. The reduction of bioactivity of TNF-α is quantified by survival of L929 cells treated with digestion reaction mixtures (soluble TNF-α in culture medium incubated with either soluble RgpA CAT −Z E or RgpA CAT −Z E immobilized in supraparticles). * indicates p ≤ 0.05. Error bars represent standard deviations (n = 3). Reprinted with permission from ref 28. Copyright 2016 The Royal Society of Chemistry.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

This Account was written through contributions of both authors. Notes

increased the viability of L929 cells over the soluble form. This indicates that the immobilized enzymes enhanced inactivation of TNF-α compared with soluble enzyme, potentially as a result of the increased stability on solid supports by prevention of autolysis. RgpA shows high specificity in proteolysis of Arg-Xaa peptide bonds,47 which occur in proteins besides TNF-α. Therefore, the high-affinity immobilization of RgpACAT−ZE within the porous supraparticles can reduce the potential risk of soluble enzymes, and the large size of the supraparticles can prohibit their internalization into most human cells. Moreover, the pores in supraparticles could partially hinder diffusion and localize the target substrate (TNF-α) in close proximity to the immobilized RgpACAT, which could increase the effective substrate concentration and degradation. In this regard, the enzyme-immobilized supraparticles have potential as carrier materials for therapeutic enzymes.

The authors declare no competing financial interest. Biographies Yeongseon Jang received her Ph.D. in Chemical and Biological Engineering under the supervision of Prof. Kookheon Char at Seoul National University. She then worked at the University of Pennsylvania as a postdoctoral researcher, coadvised by Profs. Daeyeon Lee and Daniel A. Hammer. She is currently a postdoctoral researcher in Prof. Julie Champion’s group at Georgia Institute of Technology. Her research interests include fundamental studies of the phase transition behavior of fusion proteins into self-assembled supramolecular structures and their biomedical applications. Julie A. Champion is an associate professor at Georgia Institute of Technology in the School of Chemical & Biomolecular Engineering. Her lab develops therapeutic materials, materials made directly from therapeutic proteins by self-assembly or bioconjugation, for immunomodulatory and cancer applications. She earned her Ph.D. in Chemical Engineering from the University of California, Santa Barbara in 2007 and completed a postdoctoral fellowship at California Institute of Technology in 2009.

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CONCLUSIONS AND OUTLOOK Self-assembly of materials from functional recombinant proteins with controlled physical properties and structures remains challenging. The large size, folded structure, and potential for both nonspecific and specific interactions are all contributing factors for their self-assembly. Furthermore, because of the extreme diversity of protein sequence and structure space, it can be difficult to extend knowledge of a protein material made with one functional protein to create a new material from a different functional protein. In this Account, we discussed several examples that demonstrate approaches for creating functional-protein-based self-assembled materials with diverse structures ranging from dynamic solid

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ACKNOWLEDGMENTS This work was supported by the National Science Foundation (CBET 1510551) and a kind donation from M. T. Campagna. REFERENCES

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