Protein Assembly: Versatile Approaches to Construct Highly Ordered

Review pubs.acs.org/CR

Protein Assembly: Versatile Approaches to Construct Highly Ordered Nanostructures Quan Luo,† Chunxi Hou,† Yushi Bai,† Ruibing Wang,‡ and Junqiu Liu*,† †

State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, P. R. China ‡ State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau SAR 999078, China ABSTRACT: Nature endows life with a wide variety of sophisticated, synergistic, and highly functional protein assemblies. Following Nature’s inspiration to assemble protein building blocks into exquisite nanostructures is emerging as a fascinating research field. Dictating protein assembly to obtain highly ordered nanostructures and sophisticated functions not only provides a powerful tool to understand the natural protein assembly process but also offers access to advanced biomaterials. Over the past couple of decades, the field of protein assembly has undergone unexpected and rapid developments, and various innovative strategies have been proposed. This Review outlines recent advances in the field of protein assembly and summarizes several strategies, including biotechnological strategies, chemical strategies, and combinations of these approaches, for manipulating proteins to self-assemble into desired nanostructures. The emergent applications of protein assemblies as versatile platforms to design a wide variety of attractive functional materials with improved performances have also been discussed. The goal of this Review is to highlight the importance of this highly interdisciplinary field and to promote its growth in a diverse variety of research fields ranging from nanoscience and material science to synthetic biology.

CONTENTS 1. Introduction 2. Biotechnological Strategies for Protein Assembly 2.1. Symmetry-Based Protein Assembly 2.2. Computational Design Guided Protein Assembly 2.3. Peptide-Mediated Protein Assembly 2.3.1. Amyloid-Motif-Mediated Protein Assembly 2.3.2. Coiled-Coil Motif Mediated Protein Assembly 2.4. Biological Template-Induced Protein SelfAssembly 2.4.1. DNAs as Templates 2.4.2. Proteins as Templates 2.5. In Vivo Protein Self-Assembly 3. Chemical Strategies for Protein Assembly 3.1. Receptor−Ligand Interaction-Directed Protein Assembly 3.1.1. Heme−Hemeprotein Interaction Pair 3.1.2. Lectin−Sugar Interaction Pair 3.1.3. (Strept)avidin−Biotin Interaction Pair 3.1.4. Other Interaction Pairs 3.1.5. Cooperative Receptor−Ligand Interactions 3.2. Metal-Coordination-Driven Protein Assembly

© 2016 American Chemical Society

3.3. Electrostatic-Interaction-Induced Protein Assembly 3.4. Host−Guest Recognition-Driven Protein Assembly 3.5. Polymer−Protein Conjugates Self-Assembly 3.5.1. Non-Covalently Linked Polymer−Protein Conjugates Self-Assembly 3.5.2. Covalently Linked Polymer−Protein Conjugates Self-Assembly 3.6. Chemical Template-Induced Protein Assembly 3.6.1. Nanoparticles as Templates 3.6.2. Polymers as Templates 3.6.3. Supramolecular Aggregates as Templates 3.6.4. Templated Protein Assembly via Layerby-Layer Strategy 3.7. Covalent Protein Self-Assembly 4. Applications of Protein Assemblies 4.1. Self-Assembled Protein Biosensors 4.2. Protein-Based Biocatalytic Nanomaterials 4.3. Biomedical Diagnosis and Therapy 4.4. Other Functional Materials 5. Conclusion and Outlook Author Information

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Received: April 8, 2016 Published: September 2, 2016 13571

DOI: 10.1021/acs.chemrev.6b00228 Chem. Rev. 2016, 116, 13571−13632

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Scheme 1. Schematic Representation of the Self-Assembly Strategies to Construct Highly Ordered Protein Nanostructures

Corresponding Author Notes Biographies Acknowledgments References

new generation of biomaterials. In fact, building nanostructures by assembling biomolecules in an organized way is a rapidly growing research field in nanoscience and has attracted great interest from scientists who endeavor to mimic Nature’s elegance by rational design and control of biomacromolecules to create manmade nanostructures with customized characteristics.4−6 A typical example is structural DNA nanotechnology, in which DNA oligonucleotides have been used as building blocks to create various complex and discrete nanoarchitectures via hierarchical assembly through strict Watson−Crick basepairing rules.7−11 Proteins are Nature’s extremely versatile building blocks for organisms with sophisticated topological structures and broad functions; thus, they have been considered as ideal building blocks for the “bottom-up” construction of biomolecule-based nanostructures. In comparison with simple DNA molecules, the programmable self-assembly of proteins is still at a relatively early stage of development, but this field holds great potential due to the structural complexity and functional diversity of assembled structures.12 Self-assembly of proteins into periodic arrays could create new possibilities to develop innovative bionanomaterials through combining the protein’s capabilities with a favorable microenvironment created by well-organized nanostructures.13 Moreover, understanding the self-assembly mechanism of proteins may also provide valuable insight for the treatment

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1. INTRODUCTION Natural selection and evolution have not only created a set of macromolecules to perform essential roles in biological organisms but also have endowed them with the ability to dynamically and specifically interact and assemble to yield elaborate systems of more advanced functionalities with high precision, efficiency, and adaptability.1 From well-ordered protein aggregates (e.g., viral capsids, tubulins, actin filaments, and flagella), topologically programmed nucleic acid chains (e.g., DNA duplexes, RNA triplexes, and G-quadruplexes), to more complicated nucleosomes and ribosomes, various homomeric and heteromeric complexes are formed by spontaneous and continuous assembly/disassembly in order to achieve collective properties and marvelous functions, such as genome packaging, structural support, force generation, and information storage and transmission.2,3 The spiritual wisdom of Nature offers great inspiration for the “bottom-up” fabrication of biomacromolecule-based nanostructures for a 13572


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Scheme 2. Natural Protein Assemblies with Diverse Structures for Symmetry-Dependent Biological Functions: Collagen (PDB: 3B0S); Amyloids (PDB: 2MXU); β-clamp (PDB: 2POL); TRAP (PDB: 1QAW); Bacteriophage Tail (PDB: 4JPP); αHemolysin (PDB: 7AHL); Ferritin (PDB: 1BFR) (Adapted with Permission from Ref 35; Copyright 2012 Nature Publishing Group)

high binding energies to control the thermodynamics and kinetics of the protein self-assembly process.28 By using chemical and biological technologies or a combination of these technologies, important advances have been made in the field of protein assembly. Various novel strategies, typically including symmetric fusion-based selfassembly, computational design guided self-assembly, metal ion mediated self-assembly, specific supramolecular interaction induced self-assembly, and natural/synthetic molecule templated self-assembly, have been developed to construct wellorganized protein nanoarchitectures, ranging from nanofibers, hollow tubes, ring-like structures, spherically shaped cages, to multiscale layers and crystals.19,21,29 In addition, protein engineering and site-specific chemical modification methods have greatly promoted the development of protein assembly by producing semisynthetic or hybrid proteins for specific protein interactions and for novel functionalities.30 Up to now, a variety of self-assembled nanomaterials have been fabricated by coupling highly ordered protein architectures with diverse functions of precisely positioned functional groups/molecules/ nanoparticles for a wide range of applications, such as nanodevices, biocatalysis, and drug delivery, as well as diagnosis and therapy.31 This Review outlines the recent achievements and new trends in self-assembling protein nanotechnology ranging from in vitro biomimetic protein assembly to in vivo protein assembly within actual cellular environments. Some reliable biological and chemical methods, especially supramolecular assembly of different protein monomers or geometrically

of serious human diseases, including Alzheimer’s, Parkinson’s, and other neurodegenerative diseases that are caused by abnormal protein aggregations.14,15 Nevertheless, proteins are polypeptide chains that can properly fold into particular 3D shapes via intramolecular noncovalent interactions. Conformational heterogeneity, flexibility, and complexity are important features of protein structures that are needed for sophisticated cellular functions and, in many cases, are also the major hurdles to control the order and orientation of the protein-aggregation process.16 In addition, the structural instability of proteins is affected by environmental factors such as the pH, temperature, ionic strength, and choice of solvent, which further increase the difficulty of protein manipulation.17 These disadvantages make protein self-assembly even more challenging. At the early stages of the development of this field, the exploration of protein-assembly strategies is undoubtedly the main focus point, and then the consideration of functionalities and their practical applications has gradually become emphasized.18−21 The strategies for the construction and tuning of self-assembled protein nanostructures mainly rely on an in-depth analysis and meticulous design of protein− protein interactions (PPIs), which drive protein association by biological technologies.22 However, with the development of supramolecular chemistry, chemical strategies have begun to produce many encouraging results23−26 and also have provided a wide range of extremely useful methods for fabricating protein assemblies.27 This can be accomplished by using genetically or chemically linked supramolecular self-associating elements with 13573


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specific protein oligomers to achieve increasingly diverse and complex architectures, will be discussed (Scheme 1). With the fast development of protein-assembly technology, many protein nanoarrays with well-defined structures can be easily functionalized and begin to find applications in various areas such as biosensing, drug and gene delivery, vaccine design, tissue engineering, biocatalysis, and bionanomaterials. It is intended that an overview of these protein-assembly nanotechnologies and their attractive applications will shed light on the future development of this promising research field.

2. BIOTECHNOLOGICAL STRATEGIES FOR PROTEIN ASSEMBLY 2.1. Symmetry-Based Protein Assembly

Symmetric structures are ubiquitous in biological systems because symmetric interfaces can lead to energetically favorable interactions under evolutionary stress.32 Various selective advantages have driven the self-assembly of monomeric proteins into oligomers through different domains to achieve particular symmetries, including linear, cyclic, dihedral, cubic, planar, and spatial symmetries for morphological functions and physicochemical requirements (Scheme 2).2,33−35 The relative orientation of two adjacent domains and complementary interactions within their contact interfaces define the multiplicity of oligomeric protein complexes,36,37 making it possible to design and prepare nanoscale protein assemblies in a predictable way through geometrically controlled fusion of two independent protein domains into a single larger molecule.18 In their quest to build biomimetic assemblies that rival the size and complexity of natural protein assemblies, scientists have made great efforts to design symmetric protein assemblies by biologically and chemically linking oligomeric protein domains. In this regard, some major factors that influence protein assembly need to be considered: First, the connected structural motifs should be sufficiently stable and have a strong tendency to recognize each other under the same environmental conditions. Second, natural oligomeric proteins usually have multiple symmetry operators, but only a certain axis satisfies the design requirement and can be utilized for constructing a specific geometric pattern, which may simplify the design. Third, in addition to utilizing the symmetry feature of building blocks themselves, the introduction of some mediators such as molecular linkers and small ligands allows the pre-existing interaction interfaces to be regulated by additional control to construct novel symmetric architectures. Yeates and co-workers reported the pioneering idea of a genetically oligomeric fusion method for the development of highly ordered self-assembled architectures.38 Two natural oligomeric domains, dimeric M1 matrix protein and trimeric bromoperoxidase, were held together in a predetermined orientation by genetic fusion of the two domains and with a semirigid helical linker. In this case, the degree of 3D orientation between the fusion partners can be controlled by adjusting the structural rigidity of the junction. As shown in Figure 1a, heterogeneous protein building blocks self-assembled into a 12-subunit tetrahedral cage when the specific geometric rule that the 3-fold rotational axis and the 2-fold rotational axis from each oligomeric species intersect at a proper nonorthogonal angle was adopted. Additionally, linear symmetries were obtained by replacing the trimeric fusion component with carboxylesterase, which tends to dimerize via head-to-head and tail-to-tail complexation to form protein filaments (Figure 1b).

Figure 1. Symmetry strategy to design tetrahedral (a) and linear (b) protein assemblies by genetic fusion of two distinct oligomeric proteins, bromoperoxidase and carboxylesterase, to M1 matrix protein domain. Adapted with permission from ref 38. Copyright 2001 National Academy of Sciences.

The shape, molecular mass, and size of these protein assemblies were determined via high-voltage electron microscopy (HVEM), analytic ultracentrifuge, and dynamic light scattering (DLS) characterization techniques. This finding indicates that the symmetry-based fusion strategy is incredibly effective for creating various regular protein assemblies. To resolve the cagelike structure at atomic detail, the earlier design was improved by the same group. A homogeneous 12-subunit 16nm cage with a central opening of 5 nm in diameter was obtained and crystallized after a series of mutations were conducted to avoid potential steric conflicts between protein− protein stacking interfaces.39,40 Therefore, more accurate 3D structure models can be established to guide the future design of novel protein assemblies. Using the oligomeric fusion method, a wide range of assembly architectures can be achieved, and the increasingly diverse symmetries indispensably require structurally distinct protein oligomers and are subject to different geometric rules. Yamashita and co-workers employed two symmetric protein subunits, Listeria innocua Dps protein (LisDps) and C-terminal domain of gp5 from the bacteriophage T4 (gp5C), to construct a protein assembly with a novel ball-and-spike structure (Figure 2a).41 The crystal structure shows that gp5C is a triple-stranded β-helix intertwined together to form a stable equilateral triangular prism, while LisDps is a dodecameric cagelike oligomer. Both gp5C and LisDps have a 3-fold symmetry axis and similar distances between the three N/C termini. Once gp5C was fused to the N-terminus of LisDps via a flexible peptide linker, the tubelike gp5C protein could be arrayed in a longitudinal arrangement parallel to the axis of the spherical surface of LisDps to form a ball-and-spike protein assembly. Transmission electron microscopy (TEM), dynamic light scattering (DLS), and circular dichroism (CD) results have demonstrated the formation of branched protein nanostructures with the expected diameter of 22.6 nm in solution, revealing their latent capacity for constructing advanced architectures through further hierarchical assembly. Various 3D morphologies could be realized when specific symmetric fusion design rules such as “nanohedra” were 13574


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shaped cage (Figure 3a).43 According to the helix-based oligomer-fusion design protocol, the 2-fold symmetry axis of

Figure 3. (a) Symmetric assembly of KDPGal−FkpA fusion protein into a highly porous cube-shaped cage. (b) Three major assembly forms of KDPGal−FkpA fusion proteins in solution, including 12-, 18-, and 24-subunit assemblies. Adapted with permission from ref 43. Copyright 2014 Nature Publishing Group.

FkpA was expected to be inclined at an approximate angle of 35.3° with respect to the 3-fold symmetry axis of KDPGal to achieve the geometry required for a cubic cage with an octahedral symmetry. Size-exclusion chromatography (SEC) and native polyacrylamide gel electrophoresis (PAGE) were used to determine the best linker sequence of the protein cage variants for crystallization. X-ray diffraction data confirmed that the designed cubic cage with an outer diameter of 225 Å and an inner diameter of 132 Å is the largest example reported so far. Such highly porous protein scaffolds offer unique advantages for many applications, such as macromolecule encapsulation, assisted crystallization, and X-ray analysis. In addition, native mass spectrometry (MS) found two additional protein assemblies in solution, including a 12-subunit tetrahedral assembly and an 18-subunit triangular prism assembly (Figure 3b).43 The conformational flexibility of protein building blocks may affect their assembly states, leading to a new challenge in future accuracy of architecture design. Highly sophisticated assembly architectures would be created by a combination of intrinsic structural properties, particularly through a regulated fusion orientation, and a symmetric morphology of protein components. The increased versatility of building blocks usually requires a more complicated execution. To simplify the design process, Noble and co-workers recently reported a symmetry-matching fusion method that fuses highly symmetric oligomeric proteins together with matching rotational symmetries and thereby offers numerous connection points that meet geometric design requirements.44,45 Four types of natural oligomeric proteins, including D2-symmetric streptavidin (STV)/streptagI, DsRed, D4-symmetric aminolevulinic acid dehydrogenase (ALAD), and C2-symmetric Lac21E/Lac21K, with the common structural features of N- and/or C-termini positioned close to a specific rotational symmetry axis were selected to generate extended protein arrays in one (filaments), two (molecular layers), and three (crystals) dimensions by compelling these homologous or heterologous protein subunits to align along a shared symmetry axis and leaving only one degree of freedom to control the

Figure 2. (a) Self-assembly of gp5C−LisDps fusion proteins into a ball-and-spike structure. Adapted with permission from ref 41. Copyright 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (b) Self-assembly of WA20−foldon fusion proteins into 3D nanoarchitectures using the “nanohedra” design strategy. Adapted with permission from ref 42. Copyright 2015 American Chemical Society.

adopted to the branched structural units. Arai and co-workers employed this strategy to develop a series of homooligomeric protein complexes in multiples of 6-mer based on WA20− foldon fusion protein. WA20 comprises two monomers that hold together to form an unusual 3D-domain-swapped quadruple helix bundle dimer, while the foldon domain is a small 26-residue β-propeller-like trimeric structure and can be used as a 3-arm branched junction to alter the assembly direction for extended 3D nanostructures. In this design, the connection of the WA20 and the foldon domain via a short and relatively rigid peptide linker satisfies both 2- and 3-fold symmetry constraints. Small-angle X-ray scattering (SAXS) studies showed that 6-mer and 12-mer protein assemblies with barrel- and tetrahedron-like structures, respectively, were obtained (Figure 2b).42 This indicated that symmetry considerations provide a critical control over higher-order assembly processes in a specific manner. Although the symmetry-based protein assembly strategy has been successfully applied to design relatively simple nanoarchitectures, it remains a considerable challenge to construct large and high-level protein superstructures. A specific geometric arrangement between the symmetry axes of two different oligomeric proteins is required to make the fusion design with high accuracy. Cube-shaped and hollow/porous nanostructures are one of the more attractive classes of architectures, which have been successfully prepared recently. Yeates’ group demonstrated that trimeric 2-keto-3-deoxy-6-phosphogalactonate (KDPGal) aldolase and the dimeric N-terminal domain of FkpA protein were genetically fused by a continuous α-helical linker and ultimately self-assembled into a highly porous cube13575


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2.2. Computational Design Guided Protein Assembly

relative orientation of the fusion components (Figure 4). As an important rule of this fusion design, the rigid linkers and

Large and complementary contact interfaces of natural oligomeric proteins play a fundamental role in their selfassembly processes.46 Although the controlled fusion technology has demonstrated that the connection between well-studied binding domains allows for an exploration of new classes of rationally designed protein assemblies, it remains challenging to understand and ultimately modulate their interfacial recognition behaviors in a predictive manner. With the rapid development of computational biology, many theoretical simulation methods, such as Rosetta, Z-Dock, GRAMM-X, and other commercially available programs, have been established as effective tools to guide experimental design for protein self-assembly with atomic resolution.47,48 These methods provide a wide variety of sampling algorithms to search the conformational space of proteins and scoring functions to deduce the possible binding affinities,49,50 which opens an alternative way to develop protein assemblies by redesigning the recognition specificity of existing interfaces to manipulate PPIs in order to obtain a targeted superstructure.51 The accuracies of these predictions strongly depend on the quality of the energy function and the adequate sampling of conformations. From a historical perspective, computational protein simulations were employed to investigate the ab initio protein-folding problem.52 These methods were subsequently extended to other related research fields such as computational design of protein structures,53 protein−ligand interactions,54 and even protein−protein associations.55 The lowest free energy states were identified to determine the protein recognition sites and the energetic hot spots in protein complexes for protein engineering applications.56 In contrast to the oligomer fusion approach, computational protein design is able to create completely new interfaces between naturally occurring proteins, leading to a highly diverse range of shapes. An early example of the strategy for introducing specificity into PPI was reported by Bolon et al., who reengineered an SspB homodimer into a stable SspB−ClpX heterodimer by adjusting the sequences in order to maximize the favorable interactions between ClpX and SspB.57 In view of the success of this trial, it is not surprising that computational design on many other protein systems can also achieve the dimerization process.58,59 For instance, Kuhlman and co-workers have computationally designed a symmetric homodimer via intermolecular β-sheet interactions (Figure 5).60 Rosetta’s symmetry design protocols with side-chain/backbone minimization were implemented to find an antiparallel β-strand pairing that can be engineered into protein scaffolds for interface-spanning hydrogen-bond-induced protein self-assembly. On the basis of high-quality dimeric models created by computational interface design, multiple copies of the protein subunits can be arranged in a linear fashion by integrating the designed protein−protein binding specificity and secondary intermolecular interactions on the opposite site. This strategy has been successfully applied to highly sequence-specific DNA binding proteins in the fabrication of coassembling protein− DNA nanowires. For instance, Drosophila Engrailed homeodomain (ENH) consists of three α-helices with helix 3 for sequence-specific dsDNA recognition (TAATNN). Fast Fourier transform-based docking can maximize energetically favorable interactions between two monomeric ENH proteins, which were then optimized by molecular dynamics (MD) simulations to obtain the stabilized dual-ENH with dual

Figure 4. Design principle for multidimensional protein lattices using a symmetry-matching fusion protein strategy. TEM images of 1D crysalin (DsRed-STV/Streptag I assemblies) and 2D crysalin (ALADSTV/Streptag I assemblies and ALAD-Lac21E/K assemblies). Adapted with permission from ref 45. Copyright 2011 Nature Publishing Group.

rational point group symmetry combinations are considered as structural determinants to prevent flexible or incorrect assembly. Through the careful manipulation of these assembly factors, it is possible to control the crystal growth and structure via adjustment of the relative twist angle of adjacent protein components for better compatibility with the desired lattice period. In a comparison of unmodified and modified crysalin lattices, it was found that the functionalization of protein crysalins does not influence their ability to undergo selfassembly, showing great potential to pattern a variety of substances for diverse applications in bioinorganic electrochemistry, biosensing, and biological nanocrystallography. The symmetry-based design of protein assemblies offers tremendous opportunities to develop large-scale biomimetic nanomaterials, including spatially organized protein arrays and periodic protein lattices. This approach is readily adaptable to pre-existing, well-characterized oligomeric proteins and utilizes the nature of their interfacial driving force to extend the sizes, orders, and shapes of covalently or noncovalently linked heterogeneous protein subunits. In contrast with artificially created protein interaction interfaces, naturally evolved oligomeric proteins have more stable, definite interfacial structures and binding sites, which result in a close packing arrangement of fusion protein units. Therefore, the relative orientation between neighboring building blocks and their binding stoichiometry can be precisely controlled to ensure high-level accuracy of symmetric design. On the other hand, it might also cause the possibility of unfavorable steric conflicts on the designed fusion orientation, leading to undesired assembly structures. Efforts to gain a deeper understanding of the effects of symmetric assembly factors are currently in progress, and they are driven by the need to control and tune a desired self-assembly process. The successes of various recent design efforts suggest that this strategy has promising potential to lead toward the hierarchical assembly of multicomponent protein building blocks into predictable and complex morphologies for diverse functional applications. 13576


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patterns. Baker and co-workers introduced a new design strategy that combines Rosetta design calculations with familyspecific structural constraints.62 Using RosettaRemodel, the idealized repeat units with low-energy interfaces can be generated automatically based on the available family-based sequence structural information. Very recently, this method has proven to be effective in the control of the binding surface of leucine-rich-repeat (LRR) proteins to yield a custom-designed shape.63 A series of LRR modules with variable lengths was designed in order to define the global curvature diversity. The computational design protocol optimized the relative orientations between individual LRR units and junction modules separately (Figure 7). As a result, the desired repeat-protein

Figure 5. Computational design of protein homodimer via β-strandinduced protein self-assembly. Adapted with permission from ref 60. Copyright 2011 National Academy of Sciences.

capacity of homodimerization and DNA binding. After the redesign and engineering of interfacial residues on dual-ENH, linear protein−DNA hybrid nanostructures were built by the coassembly of the computationally designed dual-ENH and double-stranded DNA (dsDNA) when the binding sites were designed in a proper position of dsDNA to facilitate a back-toback association between two dual-ENHs (Figure 6).61 The

Figure 7. Custom-specified curvature formed by modular selfassembly of the computationally designed leucine-rich-repeat proteins. Adapted with permission from ref 63. Copyright 2015 Nature Publishing Group.

curvature was obtained via self-assembly of the designed building blocks and a set of junction modules. X-ray crystallography has confirmed the atomic-level accuracy of the shape-complementary-scaffold design calculations. The growth of multidimensional protein crystals can also be mediated by computationally designed shape-complementary interfaces. Rosetta’s symmetric docking has been used by Baker and co-workers to construct large planar 2D protein lattices.64 In the design process, cyclic protein oligomers were placed into each layer group by aligning their shared symmetry axes, and sequence design calculations were subsequently performed to generate low-energy interfaces between different oligomers. Finally, the designed lattice models in layer groups P321, P4212, and P6 were optimized and selected for in vivo and in vitro experimental studies where the length of highly ordered 2D protein arrays reached up to 1 μm (Figure 8). Another type of 3D protein crystal in the P6 space group was reported by the Saven group, who designed a trimeric coiled-coil protein through a de novo approach to yield microsized polar arrays.65 Instead, a statistical thermodynamic method that integrates AMBER energy function and a discrete rotamer library were used for the sequence design calculations.

Figure 6. Coassembly of the docked ENH homodimers and dsDNA into linear supramolecular structures. Adapted with permission from ref 61. Copyright 2015 Nature Publishing Group.

structural diversity could be expanded in future work through geometric control and additional modification. However, the design of more sophisticated protein superstructures still largely relies on the atomic-level accuracy of theoretical models. Computational approaches for repeat protein design open up new possibilities for the modular assembly of the selfcompatible building blocks into large and more complex 13577


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Figure 8. Self-assembled 2D protein crystals mediated by the computationally designed protein−protein interfaces. Adapted with permission from ref 64. Copyright 2015 The American Association for the Advancement of Science.

hydrophobic surfaces of SWNTs, α-helical coiled coils were selected to realize the surface assembly process through the formation of a hexameric antiparallel protein supercoil along the SWNT axis. Interestingly, two-dimensional photoluminescence (2D-PL) spectra showed that the protein assemblies formed by HexCoil-Gly and HexCoil-Ala sequences had identical chirality to that of SWNTs. Recently, a significant contribution was made by Baker and co-workers, who successfully prepared two types of cagelike protein assemblies to mimic virus capsid structures. A computational method that combines symmetrical docking and protein−protein interface design was developed to aid the design of protein assemblies with a specific point group symmetry. In the first case, C3-symmetric protein trimers were chosen as building blocks for symmetric docking. The 3-fold axis of each building block was aligned along the direction of the tetrahedral or octahedral 3-fold axis of the desired architectures, and then sequence design was performed by RosettaDesign calculations in order to create low-energy, symmetric PPIs. As expected, the designed interface reached the binding strength of naturally occurring oligomeric proteins and, therefore, drove the self-assembly of trimeric protein units into synthetic supramolecular nanocages, a 24-subunit cage with octahedral symmetry and a 12-subunit cage with a tetrahedral symmetry (Figure 10a).68 A further attempt on this strategy was through the use of two distinct protein subunits for coassembly studies in which the arrangement of the symmetry axes of heterogeneous trimeric or dimeric species must conform perfectly to the requirements of tetrahedral point group symmetry, and the designed interfaces provided a sufficiently strong driving force to control the binding orientation and specificity of the building blocks so as to construct dual tetrahedral architectures (Figure 10b).69 These studies demonstrated the capability of computational simulation to design new oligomeric protein structures with atomic-level accuracy through controllable assembly and, therefore, should open up new opportunities for the development of targeted drug delivery, vaccine design, plasmonics, and other applications that can benefit from high-precision patterning at the nanoscale. Computational and theoretical studies provide insights into the structural and mechanistic details of PPIs that are important for guiding and controlling the self-assembly process through

Viruses and virus-like particles (VLPs) represent one of the most exquisite architectures created by Nature.66 Computational design of such hierarchically ordered nanostructures needs to consider not only the protein−protein interaction specificity but also the point group symmetry. Grigoryan et al. pioneered the design of virus-like protein assemblies on the surfaces of single-walled carbon nanotubes (SWNTs) by utilizing their surface properties and symmetry features (Figure 9).67 Three rules have been proposed in the calculation process to design the peptide models for symmetry-matching regulated protein−SWNTs interactions, including the identification of compatible groups for the target surface, the definition of peptide superstructures on the target surface, and the determination of the designability of new protein−protein interfaces. On the basis of the cylindrical shape and

Figure 9. Computational design protocol to construct virus-like protein assemblies on carbon nanotube surfaces. Adapted with permission from ref 67. Copyright 2011 The American Association for the Advancement of Science. 13578


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and extended ordered arrays.74 In fact, self-assembled peptide nanomaterials have been discussed in many reviews6,75,76 and are beyond the scope of this section. Here, we only address smaller protein-motif-mediated protein-assembly processes, including those involving amyloid and coiled-coil motifs. 2.3.1. Amyloid-Motif-Mediated Protein Assembly. In living systems, β-strands are the key components in the formation of highly stable fibrillar structures via intramolecular hydrogen bonding and the hydrophobic interactions due to their inherently gregarious nature.77,78 The most-studied example is the amyloid β (Aβ), a peptide of 36−43 amino acids that can self-assemble into insoluble β-sheet-rich amyloid fibrils to cause Alzheimer’s and other neurodegenerative diseases.79 Several studies have indicated that Aβ has evolved specific amyloidogenic core sequences of 4−7 amino acids and a highly ordered cross-β structure to maximize the stability between both side chain and backbone atoms during the selfassembly process.80 On the basis of the redesign and modification of the amyloidogenic sequences, a variety of methods (e.g., combinatorial library and halogenation) have been developed to tune their self-assembly behavior to yield distinct fibrillar morphologies.81−85 As researchers have gained a deeper understanding of the relationship between amino acid sequences and the propensity to form amyloids, the development of β-sheet amyloid mimics (BAMs) has attracted much attention.86 Eisenberg and coworkers reported a rare out-of-register amyloid-like fibril formed by de novo designed β-sheet macrocycles that contain two antiparallel β-strands covalently connected by a δ-linked ornithine turn.87 Each strand has the ability to recognize another corresponding strand through intermolecular hydrogen bonding. As shown in Figure 11a, the “upper” strands consist of

Figure 10. Computationally designed cagelike protein assemblies with octahedral, tetrahedral (a), or dual tetrahedral (b) point group symmetries. Adapted with permission from ref 68. Copyright 2012 The American Association for the Advancement of Science. Adapted with permission from ref 69. Copyright 2014 Nature Publishing Group.

design and engineering of interfacial sequence motifs with low binding energies. The de novo design of completely new interaction surfaces allows greater structural complexity and diversity, which have been shown to be highly valuable in developing advanced protein nanostructures with superior rigidity and monodispersity (e.g., virus-like structures) typically found in Nature.70 Although new algorithms have facilitated the rapid development of this field and made it possible to transfer nonself-associating proteins into self-associating proteins through a minimal number of mutations, the existing computational capabilities are still limited, and thus, it is difficult to achieve a good balance between computational accuracy and efficiency for large-scale protein assembly systems. As a long-term perspective, more accurate and complete PPI networks are expected to be established from computationally derived interaction data sets, which is a crucial step for further exploration of the mechanism, regulation, and complexity of protein assembly. 2.3. Peptide-Mediated Protein Assembly

Individual secondary structures such as α-helices and β-sheets are critical elements of oligomeric protein interfaces.71 These specific motifs are short peptide folding patterns with a highly characteristic sequence that acts cooperatively to determine the interaction specificity between both naturally occurring and computationally designed protein subunits.72 Although the isolated structural motifs alone are usually too weak to achieve high affinity to one another, a bundle of them wrapped together often exhibits a sufficiently strong driving force beyond a single peptide sequence to drive peptide-induced protein association.73 The complexity of the assembled protein nanostructures arises from the variable sequences and quantified components that govern the geometrically precise interactions to form a variety of aggregation morphologies such as filaments, cages,

Figure 11. Amyloid-like fibrils formed by self-assembly of de novo designed β-sheet macrocycles. (a) Structure of macrocyclic peptide; (b) out-of-register β-strands. Adapted with permission from ref 87. Copyright 2012 National Academy of Sciences. 13579


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combinatorial flexibility for structural and functional improvement of their self-assembly studies by controllable integration of multiple different bioactive components together. Recent efforts on the design and assembly of combinatorial β-sheet motifs have made dramatic achievements in biomimetic materials. The minimal virus-like protein assemblies have successfully demonstrated the precision of this method to control the cooperativity of multifold-mediated self-assembly of C−Sn−B proteins (C, C-terminal 407-amino-acid hydrophilic random coil; Sn, n-fold repetition of a silklike sequence; B, Nterminal dodecalysine for DNA binding) with single DNA molecules for a rod-shaped virus-like morphology (Figure 13a).93 In addition, amyloid-like nanostructures can be

heptapeptide sequences extracted from various native amyloid proteins for an antiparallel β-interaction between two “upper” strands, while the “lower” strands contain a tripeptide mimic (Hao) and two flanking dipeptides to stabilize the βconformation. The structural design of the unnatural amino acid Hao is able to partially block the hydrogen-bonding network between adjacent “lower” strands, leading to an out of register arrangement of β-strands (Figure 11b). Notably, amyloid-like β-sheets and their derivatives can be further tailored to inhibit the aggregation of amyloid proteins, which shows great potential in the future treatment of amyloid-related diseases.88,89 Two and more β-sheets associated in the lateral direction could produce more sophisticated protein assemblies. Matsuura et al. reported the first example of a spherical hollow protein assembly formed by a 24-mer β-annulus peptide from tomato bushy stunt virus (TBSV) capsids (Figure 12a).90 This peptide

Figure 13. Biomimetic materials made by the self-assembly of combinatorial β-sheet motifs, including (a) virus-like coat proteins and (b) underwater adhesive fibers. Adapted with permission from refs 93 and 94. Copyright 2014 Nature Publishing Group.

Figure 12. Variety of sophisticated morphologies formed by selfassembly of multiple β-sheet motifs, including (a) a nanocapsule, (b) a nanotube, and (c) a nanofiber. Adapted with permission from refs 90, 91, and 92. Copyright 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim (refs 90 and 91), and 2014 Nature Publishing Group (ref 92).

designed to mimic and improve naturally occurring underwater adhesive based on adhesive protein domains, mussel foot proteins (Mfps), and CsgA proteins. Using a combinatorial genetic strategy, two fusion proteins, CsgA−Mfp3 and Mfp5− CsgA, were constructed and then self-assembled into hybrid fibrous materials, where the CsgA domain has a cross-β structure and can self-polymerize to form the amyloid core with unstructured Mfps displayed on the surface (Figure 13b).94 Colloid probe atomic force microscopy (AFM) studies showed that the resulting biomimetic fibers exhibited strong underwater adhesiveness. These findings may open up new opportunities for exploration of bioinspired materials by incorporation of a diverse library of functional biomolecules into amyloid-based assemblies. 2.3.2. Coiled-Coil Motif Mediated Protein Assembly. Coiled coils are another important type of structural motif involved in protein oligomerization.95 Since Astbury discovered α-form from natural fibers by X-ray diffraction in the 1930s, numerous studies have been carried out to ascertain its structure, properties, and functions.96−98 Coiled coils were

fragment was found to have three β-sheet motifs and could selfassemble into a C3-symmetric structure in which equivalent pieces of β-strand wrap around a 3-fold axis to form a unique “β-annulus” structure. The strong interactions between the sticky ends of the β-annulus structures induced further aggregation of the trigonal β-sheet-forming peptides to form virus-like nanocapsules in water. Furthermore, the design of multiple β-sheet motifs connected by covalent or noncovalent methods have also contributed to the morphological diversity of protein assemblies. The groups of Ueno and Collier have expanded the possibilities for constructing a robust bionanotube and nanofiber based on the dimerization of a triplestranded β-helix of (gp5βf)3 and the gradated assembly of different βTail fusion proteins, respectively (Figure 12b, c).91,92 These studies suggested that β-sheet motifs have great 13580


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characterized as heptad repeats with the recurring “HPPHCPC” sequence of hydrophobic (H), polar (P), and charged (C) amino acid residues, where hydrophobic residues come together to form an amphipathic structure for driving and modulating protein aggregation through a “knobs-into-holes” packing arrangement.99 Inspired by the sequence and structural features of well-studied oligomeric protein domains,100−102 various de novo designed coiled coils have been synthesized to study and mimic PPIs. The structural stability, strand orientation, oligomerization degree, and selectivity of these αhelical motifs are varied by changing their lengths, hydrophilic/ hydrophobic residue distributions, and connection methods. Dimeric coiled coils are the most common oligomerization state and have been investigated extensively.103,104 Kwok and Hodges designed a series of homodimeric parallel coiled coils and demonstrated that the increased chain length can improve the overall stability.105 A substitution of identically charged residues at positions e and g of each heptad repeat with oppositely charged residues could lead to the formation of heterodimers, and the orientation of two helices in a coiled coil was further mediated by interchain electrostatic interactions between residues at the two positions of the matching heptads. On the basis of this design principle, Oakley and co-workers successfully designed an antiparallel coiled-coil heterodimer by a favorable combination of hydrophilic and hydrophobic residues to affect the interhelical hydrophobic and Coulombic interactions.106 Larger-scale protein superstructures can be arranged when heterodimeric coiled coils are used as a starting point for the programmable self-assembly of fibrous biomaterials. For instance, Woolfson and co-workers constructed a parallel, dimeric coiled-coil fiber using two complementary 28residue polypeptides.107 The four heptad sequence repeats contain a “sticky end” that is a helix extending two heptad repeats beyond the main coiled-coil region for end-to-end interaction with another peptide. This allowed many α-helices to twist around each other in their longitudinal direction to form a long coiled-coil fiber. The packing geometries of individual α-helices within the fibers were clearly visualized by cryo-TEM, which provided structural insight into the assembly processes of coiled coils to form fibrous structures (Figure 14).108 Various dimeric coiled-coil domains can be genetically engineered into the protein sequence to induce protein dimerization in vitro and in vivo for different biochemical properties and functional demands. Early attempts were performed on the heterodimerization of different functional proteins such as leucine zipper protein spacer modified green fluorescent protein (GFP)/cytoplasmic structural protein and antibody heavy chain/light chain.109,110 Subsequently, a de novo designed E5/K5 coiled coil was successfully utilized to induce the homo- and heterodimerization of receptor ectodomain for a higher antagonistic potency to TGF-β1 as compared with the monomeric ectodomain.111 ProP can be activated at low osmolality when homodimeric, antiparallel coiled coil was formed in vivo at the C-terminus of ProP to stabilize its active conformation.112 This dimerization strategy was also used to design a turn-on protease biosensor by fusing two distinct coiled-coil pairs (EE/RR pair and acid/base pair) to either the N- or C-termini of split-luciferase reporter to control the catalytic activity of luciferase by protease-mediated cleavage (Figure 15a).113 A larger-scale linear protein assembly was developed by Rief and co-workers, who employed two coiled-coil binding partners to control the programmed self-

Figure 14. De novo design of heterodimeric coiled coils to construct a large protein fiber via sticky-end assembly. Adapted with permission from ref 108. Copyright 2012 National Academy of Sciences.

assembly of Ig27 and GFP proteins into linear polyprotein chains (Figure 15b).114 AFM characterization provided a direct evaluation of the mechanical properties of coiled-coil interactions by stretching single polyprotein molecules. Moreover, the dimerized coiled coils can be designed as hydrophilic blocks to construct recombinant protein amphiphiles for thermally triggered self-assembly. The arginine-rich leucine zipper motif (ZR) and glutamic acid-rich leucine zipper motif (ZE) were used to prepare three diblock fusion proteins, ZRELP, mCherry-ZE, and EGFP-ZE, which can self-assemble into hollow vesicles by mixing them together at 4 °C (Figure 15c),115 because the strong heterodimeric ZE/ZR interactions and weak homodimeric ZR/ZR interactions lead to two types of protein amphiphiles, (mCherry or EGFP)-ZE/ZR-ELP and ELP-ZR/ZR-ELP. Interestingly, due to the unique temperatureresponsive phase-transition behavior of elastin-like polypeptide (ELP), ELP-ZR/ZR-ELP has become a versatile mediator for vesicle formation, displaying great potential for applications in drug encapsulation and delivery. More complex assemblies were developed by sequential combination of multiple α-helical units to define the desired morphologies. Two distinct cage-like structures assembled from covalently linked coiled coils have demonstrated the potential of this strategy for mimicking evolutionary diversification.116 Jerala and co-workers used coiled-coil interactions to develop a 10-nm-sized monomeric tetrahedral nanocage (Figure 16a).117 A single-chain polypeptide was designed by connection of 12 concatenated coiled-coil-forming segments with short, flexible Ser-Gly-Pro-Gly peptide linkers. The self-assembly and folding processes driven by specific pairwise interactions between different coiled-coil elements allow for creation of a tetrahedral 13581


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Figure 16. Tetrahedral (a) and cagelike structures (b) formed by selfassembly of sequentially connected coiled-coil segments. Adapted with permission from ref 117. Copyright 2013 Nature Publishing Group. Adapted with permission from ref 118. Copyright 2013 The American Association for the Advancement of Science.

understand the self-assembly processes of cellular compartments and will pave the way for the development of new materials for the delivery of drugs and the development of protocells. Amyloid-like β-sheets and coiled coils represent the simplified protein models for self-assembly studies. They can also be used as hubs and spacers to hold protein components together with defined stoichiometries and orientations through genetic fusion. The variability of 20 naturally occurring amino acids in monomeric units allows for an extremely high combinatorial diversity that encodes a wide variety of peptide architectures and topologies such as parallel, antiparallel, and heterotypic β-sheets or helical bundles. The versatility and high binding specificity of these structural motifs allow peptideinduced protein assemblies to be easily extended and customized. Furthermore, the delivered design rules and computer algorithms have ensured the success of this strategy to achieve a predictable assembly. Sequence-structure manipulations and combinational arrangements are two critical considerations for the control of peptide-mediated supramolecular structures. Because the most well-known rules are formulated on the basis of the heptad repeats of the natural proteins, considerable efforts should be devoted to complementing the design rules by comparing the sequences of different natural peptide folding motifs. These findings will provide fundamental insights into the de novo design principles, assembly, and regulation mechanisms of proteinaggregation processes.

Figure 15. (a) Dimerization of split-luciferase reporter achieved by genetic engineering with coiled coils to design a turn-on protease biosensor. (b) Linear polyprotein chains formed by coiled-coilmediated protein self-assembly. (c) Coiled-coil-induced self-assembly of protein amphiphiles into vesicles. Adapted with permission from refs 113, 114, and 115. Copyright 2009 American Chemical Society, 2007 IOP Publishing, and 2014 American Chemical Society, respectively.

topology that was precisely controlled by the orientation and sequential arrangement of each coiled-coil pair. In addition, the rational design of coiled-coil modules with a uniform curvature may ensure that the geometric architecture closely matches the targeted spherical shape. A large hollow protein nanocage was reported by Woolfson et al., who engineered two fused coiled coils, CC-Tri3-CC-Di-A and CC-Tri3-CC-Di-B, via disulfide bonds for hierarchical self-assembly. Due to the complementary nature of homotrimeric CC-Tri3 and heterodimeric CC-Di-A or B, the two covalently linked modules can self-assemble into trimeric hubs, which further coassemble into hexagonal networks to form the 100-nm 3D particles by optimizing the hub−hub angle to satisfy a specific geometric constraint (Figure 16b).118 In view of the different sizes and structures of these artificial protein cages, it can be concluded that the modular organization of multiple coiled coils can serve to modulate their self-assembled architectures in a predictable manner, leading to structural diversity and complexity. The de novo design of these self-assembled cagelike structures will help us to further

2.4. Biological Template-Induced Protein Self-Assembly

The evolution in biomaterials to achieve intricate morphologies is regarded as the limit of exquisite control over molecular 13582


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aggregation processes. A cooperative interplay of multiple, weak noncovalent interactions between biomacromolecules contributes to their structural and geometric diversity based on extremely precise pairing rules. Taking advantage of the selfassembling nature of these molecules (e.g., DNA and protein) for organizing proteins represents an effective and effortless way to achieve a high level of accuracy and complexity for nanomanipulation. This strategy will provide a facile method to drive protein assembly by integrating target proteins into template molecules for programmable and periodic arrangements. 2.4.1. DNAs as Templates. DNA nanotechnology for protein self-assembly was first proposed by Seeman, who suggested that DNA nanolattices could serve as templates to organize proteins into 3D crystals for studying their structure by X-ray crystallography.119 Since then, this research field has developed rapidly and outgrown its original purpose. For instance, branched DNA junctions with sticky ends were used for the precise spatial arrangement of different functional species.7,9,120 On the basis of DNA’s structural features and Watson−Crick base-pairing rules, early attempts to build DNA nanostructures have yielded elaborate architectures that were held together by various junctions, which are too flexible to achieve structural control for large-scale nanofabrication.121,122 The subsequently developed crossover DNA tiles such as double-crossover molecules (DX), triple crossover molecules (TX), 2-, 4-, and 6-helix bundles, and cross-shaped tiles can significantly improve the structural rigidity and self-assemble into higher-order superstructures with well-defined periodicities and complexities,123 thus providing reliable frameworks to pattern proteins in elaborate designs by covalent or noncovalent attachment of them to DNA origami nanoarrays. Ligand−protein interactions are often utilized to explore DNA-templated protein self-assembly. DNA strands can be modified with a ligand molecule that has a strong and specific binding capability to its target proteins for selective immobilization.124 This method was initially proposed by Niemeyer et al., who has exploited the biotin−STV interaction to precisely position the biotinylated immunoglobulin G (IgG) or alkaline phosphatase (AP) onto the immobilized DNA arrays using the complementary DNA−STV hybrid molecules.125 In principle, almost any type of biotinylated protein can be arranged onto custom-designed DNA scaffolds by means of supramolecular bioconjugation. For example, the specific binding between N-terminal biotin-labeled recombinant enzymes (NAD(P)H:FMN oxidoreductase and luciferase) and two different DNA−STV cross-linking molecules (SA and SB) led to the formation of enzyme−DNA conjugates, which further self-assembled onto microplates through DNA− DNA hybridization to construct multienzyme complexes for synergistic catalysis (Figure 17).126 Moreover, other specific interactions such as nickel-mediated hexahistidine interactions and antibody−hapten interactions were also employed to produce protein−DNA conjugates that allowed a large number of functional proteins to be displayed on DNA-coated surfaces in a highly programmable manner.127,128 Another method for DNA-templated protein assembly is based on aptamer binding to various target proteins. DNA or RNA aptamers were engineered into DNA tiles and acted as recognition sites for site-specific protein displays. Yan and coworkers were the first to demonstrate that a DNA aptamer with a stable G-quadruplex structure was capable of selectively capturing and directing the assembly of thrombin at periodic

Figure 17. DNA-templated self-assembly of multienzyme complexes, NAD(P)H:FMN oxidoreductase (NFOR) and luciferase (Luc), through biotin−STV interactions. Adapted with permission from ref 126. Copyright 2002 Wiley-VCH Verlag GmbH, Weinheim, Germany.

sites of linear TX tile arrays (Figure 18a).129 Kenan and coworkers successfully applied this method to develop new

Figure 18. (a) Self-assembly of proteins on aptamer-containing DNA nanostructures by selective capture of thrombins. Adapted with permission from ref 129. Copyright 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (b) Supramolecular linear or branched nanostructures formed by aptamer−thrombin association. Adapted with permission from ref 132. Copyright 2008 Royal Society of Chemistry.

protein assemblies by decorating three different DNA tile-based templates with specific aptamers for organizing single-chain variable fragment (scFv) into specific patterns.130 Because the designed DNA aptamers can be used as targets for screening phage display libraries to identify specific aptamer−protein pairs for nanoscale construction, this technology has the potential to assemble a wide range of proteins and shows great 13583


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protein pathways at local concentrations reminiscent of cellular pathways. Additionally, single-strand DNA binding protein (SSB) was also used as a bifunctional linker to bridge both single-strand DNA (ssDNA) and target proteins. For example, SSB-tagged superfolder green fluorescent protein (sfGFP) allowed the development of an active protein layer when SSB bound tightly to an ssDNA-labeled matrix to realize ssDNAmediated protein immobilization. This surface binding was confirmed to be reversible and regulated by pH, divalent cation concentration, and the complementary oligonucleotide.134 An altered binding specificity between a viral capsid and its genome (RNA or DNA molecule) can also provide novel applications in protein nanofabrication. It is well-known that virus particles are able to pack a nucleic acid spontaneously during in vitro and in vivo self-assembly. Many studies have confirmed that the interactions between a coat protein (CP) and nucleotide basis are involved in the energetically and kinetically favorable folding process of the nucleic acid chain.135,136 Taking advantage of nonspecific CP−DNA binding, Zlotnick and co-workers synthesized a dsDNA to convert an icosahedral plant virus, cowpea chlorotic mottle virus (CCMV), into a tubular nanostructure (Figure 20), whose

promise for specifically targeted detection and therapy. Instead, some DNA-binding proteins such as zinc-finger proteins that can recognize specific sequences were selected for targeting specific locations within DNA-origami structures.131 In addition to incorporating the aptamers into the self-assembled DNA nanostructures, the aptamer−protein pairs themselves are versatile connectors in the construction of protein assemblies. Willner’s group designed a bisaptamer that contains a βaptamer at the 3′ end and an α-aptamer at the 5′ end for 1:2 binding ratio to thrombin. Supramolecular linear or branched protein−DNA hybrid polymers were constructed by the association of thrombins with the bidentate aptamers or the mixtures of bidentate aptamers and tripodal tridentate αaptamers (Figure 18b).132 The third method is to attach a peptide/protein to a singlestranded DNA oligonucleotide in order to facilitate site-specific protein immobilization. Williams et al. reported a DNA− peptide fusion formed by coupling the 4-(N-maleimidomethyl)cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC)-modified oligonucleotide to a cysteine residue of myc-peptide in order to place it at a specific position on a DX tile.133 The resulting DX tile combined with three other DX tiles can further assemble into a periodic 2D array with SMCCmodified tiles displaying a unique binding specificity to antimyc antibodies (Figure 19). Using this technology, tailor-made

Figure 20. DNA-templated self-assembly of CCMV coat proteins into a tubular nanostructure by altering the binding specificity between the viral capsid and DNA. Adapted with permission from ref 137. Copyright 2006 American Chemical Society.

diameter and length were affected by the geometry of the CP and the ratio of CP/DNA, respectively.137 Recently, a similar protein nanostructure was obtained by stabilizing the hexamer geometry of a CP when CCMV was self-assembled into kinetically trapped tubes on dsDNA scaffold at low pH.138 This method was found to be applicable to other spherical plant viruses, such as cucumber mosaic virus (CMV) capsid protein. In the presence of heterogeneous dsDNA templates, CMV CPs can self-assemble into highly uniform nanotubes with a diameter of only ∼17 nm.139 In fact, it will be possible to design new and more elaborate protein nanostructures through a combination of protein engineering and DNA templates in the near future. Covalent linkage of a protein molecule to a defined DNA oligonucleotide is another reliable method to yield the desired conjugates for DNA-directed protein self-assembly. The synthetic process is usually achieved by direct chemical reactions with thiol, amino, or carboxyl groups of proteins, while the self-assembling DNA oligonucleotides enable the

Figure 19. Self-assembled DNA−peptide nanoarrays for periodic arrangement of antibodies based on the binding specificity between peptides and antibodies. Adapted with permission from ref 133. Copyright 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

protein nanoarrays can be produced at addressable locations, which allows for the detection of protein binding in solution at the single-molecule level. Thus, it might provide a convenient method to study protein−protein interactions for nanobiotechnological and nanoelectronic applications, such as the fabrication of nanoelectronics and nanobiochips to monitor 13584


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antibodies) on the self-assembled DNA nanostructures (Figure 21b).144,145 Using the above-mentioned methods to localize proteins, the structural complexity of multiprotein nanoarrays depends on the DNA sequences and the programmability of the sticky ends that define the connectivity between individual DNA tiles to determine the size, periodicity, and addressability of DNA nanostructures. To construct 1D protein assemblies, the linear DNA template with numerous repeated sequences was synthesized by rolling-circle amplification (RCA) to periodically arrange proteins. Willner’s group reported two protein nanowires that were constructed by incorporating periodic aptamer sequences into linear DNA chains for selective recognition of thrombin, lysozyme, or thrombin/lysozyme to create 1D protein−DNA composites (Figure 22a).146 The final

controlled organization of proteins into multicomponent clusters or well-defined nanoarrays.140 Niemeyer and coworkers reported the synthesis of six ssDNA-fluorescent protein (FP) conjugates via a thiol-maleimide “click” reaction that immobilized them to DNA−AuNPs’ (NP = nanoparticle) surface through specific DNA hybridization.141 Recently, the thiol-selective coupling method was successfully applied to prepare Fab fragment−DNA conjugates for protein heterodimerization on a DNA-functionalized membrane surface by heterospecific DNA hybridization (Figure 21a).142 This method

Figure 21. (a) Heterodimerization of Fab′−DNA conjugates on a DNA-functionalized membrane via a thiol-selective coupling reaction and DNA hybridization. Adapted with permission from ref 142. Copyright 2013 American Chemical Society. (b) Self-assembly of ssDNA-modified virus capsids onto a DNA nanostructure with multiple complementary strands. Adapted with permission from ref 144. Copyright 2010 American Chemical Society.

Figure 22. (a) Linear DNA template with periodic aptamer sequences generated by RCA to pattern different proteins in the formation of 1D nanowires through selective recognition. Adapted with permission from ref 146. Copyright 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. (b) TX DNA-templated self-assembly of linear STV arrays based on biotin−STV interactions. Adapted with permission from ref 147. Copyright 2004 American Chemical Society.

can also be extended to anchor proteins onto live cell surfaces to regulate cell−cell signaling interactions for therapeutic purposes. An analogous selectivity was also observed for other amino acids such as lysine, in the case of a DNA−enzyme assembly, where the active lysine site of glucose oxidase (GOx) or horseradish peroxidase (HRP) was used to conjugate the alkylthiol-modified DNA oligonucleotides in order to construct a multienzyme system with controllable activity regulated by DNA hybridization/dehybridization.143 Furthermore, unnatural amino acids such as p-aminophenylalanine (pAF) and pacetylphenylalanine (pAcF) can be site-specifically incorporated onto the external surfaces of protein molecules to create a reactive group (e.g., amino and keto group); this reactive group can then react with the phenylenediamine- or aminooxymodified DNA for patterning proteins (e.g., viral capsids and

arrangement of each protein on the ssDNA scaffold can be controlled by adjusting the density of aptamers and the length of spacer between two aptamer units. In addition, more rigid protein nanowires can be constructed by using the biotinmodified TX tiles that have three sticky ends to pair with each other to form linear DNA arrays for the programmed organization of the STV proteins through biotin−STV interactions (Figure 22b).147 The design of branched DNA tiles as multiarm junctions to create noncovalently cross-linked DNA networks can extend 13585


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including tetrahedron (TET)-, octahedron (OCT)-, and icosahedron (ICO)-shaped DNA segments, were constructed by Mao and co-workers, who synthesized three different DNA strands (a long strand Ln, a medium strand M, and a short strand S) in order to prepare a family of star-shaped DNA nanomotifs for self-assembly into the desired 3D architectures (Figure 24a).152 Biotin molecules were modified at the 5′ end

the protein assemblies into 2D nanoarrays. The LaBean group pioneered the programming of the sticky-end associations of biotin-labeled 4 × 4 tiles to generate DNA nanogrids with periodic square cavities for templating STV proteins into a single flat layer (Figure 23a).148 An obvious advantage of the

Figure 23. (a) Self-assembly of protein arrays templated by 4 × 4 DNA nanogrids via biotin−STV interactions. Adapted with permission from ref 148. Copyright 2003 The American Association for the Advancement of Science. (b) S-shaped thrombin arrays on selfassembled DNA tile scaffold via biotin−STV interactions. Adapted with permission from ref 150. Copyright 2007 American Chemical Society.

Figure 24. DNA-directed 3D protein organization (a) and crystallization (b) through biotin−STV interactions and DNA−DNA interactions, respectively. Adapted with permission from ref 152. Copyright 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Adapted with permission from ref 154. Copyright 2015 National Academy of Sciences.

multitile system is that random combinatorial DNA nanostructures could be built by varying the sticky ends to enable the development of more sophisticated protein arrays. Numerous recent reports have supported the successful use of DNA tilebased self-assembly strategy to construct a variety of 2D architectures.123,149 For instance, an S-shaped protein pattern can be designed on a flat rectangular DNA scaffold that was formed by four DX tiles with one of them decorated with thrombin aptamers to achieve the precise addressability of DNA template (Figure 23b).150 Not only crossover DNA tiles but also multihelix DNA tiles composed of either four-helix bundles or five-helix bundles can self-assemble into such rigid rectangular-shaped DNA-origami structures.151 Heterobivalent aptamers were introduced into the ends of the helices and adjusted at different distances for arrangement of thrombins with nanometer-scale accuracy to study their distance-dependent multivalent binding effects. DNA-directed 3D protein assembly is one of the most difficult design challenges. A general strategy to fabricate the DNA-origami frames often involves careful consideration of both structural stability and flexibility. By maximizing the base pairing between complementary DNA segments, short single DNA strands are used to direct the folding of a long single DNA strand into symmetric nanomotifs that further assemble into DNA polyhedra, where rigid DNA duplexes serve as polyhedral wire edges and flexible branch points serve as vertexes. As a proof-of-concept, a series of polyhedral DNA,

of the S strand to spatially control the position of STV proteins on each DNA polyhedral face. Similarly, a 3D DNA tetrahedron assembled from four partially complementary oligonucleotides was used as a scaffold for site-selective orthogonal protein coupling based on strong ligand−protein interactions.153 Moreover, DNA-programmable 3D protein crystallization was induced when protein molecules were covalently conjugated with the radially oriented DNA oligonucleotides to allow their controlled self-assembly into different crystalline states through DNA−DNA interactions. Mirkin and co-workers reported the application of a copper-free azide−dibenzocyclooctyne “click” reaction to construct two DNA-modified catalases that can self-assemble into bodycentered cubic or cesium-chloride-type superlattices by hybridization with the complementary DNA linkers (Figure 24b).154 The structural and chemical heterogeneity of protein units were found to have a significant influence on the final superlattice type. 2.4.2. Proteins as Templates. Naturally occurring protein assemblies, ranging from regularly spaced 2D protein layers to 3D viruses and VLPs, provide a wide diversity of frameworks for templating nonself-associating heterologous proteins into 13586


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multidimensional arrays.155 Although these supporting scaffolds have highly intricate architectures, they can be easily manipulated and functionalized due to their genetically directed synthesis. Using the recombinant DNA techniques, passenger proteins were fused to the N- or C-terminus of carrier proteins and then self-assembled into diverse supramolecular structures by combining the intrinsic self-assembly properties of carrier proteins and their ability to display multiple copies of the fusion partners. Since the bacteriophage was first investigated to express the target proteins (e.g., enzymes, receptors, and antibodies) as fusions with phage coat proteins for surface displays, various bacterial surface display systems have been developed to anchor foreign proteins onto the exterior surface of different host cells, such as bacteria, yeasts, and fungi.156,157 As the predominant component of cell-surface structures, S-layer proteins are composed of proteins or glycoprotein species, which have the unique feature of self-assembling into 2D crystalline arrays that coat the entire bacterial cell and allow for the high-density display of large protein fragments in many nanobiotechnological applications.158 Sára and co-workers described the in vitro self-assembly of S-layer-streptavidin fusion proteins into highly ordered and oriented 2D protein crystals that were used as a template to pattern the biotinylated target proteins based on biotin−STV interactions.159 Two types of fusion proteins were constructed by creation of a fusion hybrid between STV and N- or C-terminus of the S-layer protein SbsB, in which only N-terminal fusion proteins were able to self-assemble into flat sheets both in suspension and on different supporting substrates due to the C-terminal domain of SbsB responsible for lattice formation. Efficient surface exposure of STV at the outer S-layer face has created numerous biotin-binding sites for periodic arrangement of the biotinylated ferritin, peroxidase, and two marker proteins (Figure 25a), revealing that the S-layer is an excellent template with reliable compatibility and extensibility for 2D monomolecular protein nanofabrication. This was also demonstrated by the fusion of several other functional proteins such as immunoglobulin G-binding domain, IHNV glycoprotein, fluorescent protein, methyl parathion hydrolase (MPH), and specific antigens that were shown to be exposed on the S-layer lattice to yield biologically active protein monolayers for designing novel biosensors, vaccines, biomarkers, biocatalysts, and biodetectors.160,161 Among them, dual-functionalized S-layer protein nanostructures with MPH and anthrax-specific antibodies exhibited not only improved enzymatic stability but also significantly higher anthraxdetection sensitivity than that of the unassembled complexes for multifunctional applications in nanocatalysis and biosensing (Figure 25b).161 The symmetric quasicrystal structure and high-level gene expression make viruses and VLPs attractive candidates for 3D displaying identical copies of foreign proteins. Viral capsids were genetically engineered to express fusion proteins without interfering with the nucleation and growth of CPs to form the hybrid protein superstructures. Following a breakthrough in heterologous peptides presentation on tobacco mosaic virus (TMV), cowpea mosaic virus (CPMV), and alfalfa mosaic virus (AIMV) particles,162−164 a highly oriented self-assembly of GFP was initially attempted by using the 2A oligopeptide as a connector for potato virus X (PVX)−GFP fusion to construct paracrystalline rodlike structures.165 Similarly, several other display systems, including adeno-associated virus (AAV), multiple nuclear polyhedrosis virus (MNPV), and tobacco

Figure 25. (a) Self-assembly of the biotinylated ferritin into 2D arrays in suspension, on liposomes, and on cell-wall fragments using S-layer display technology. Adapted with permission from ref 159. Copyright 2002 National Academy of Sciences. (b) Templated self-assembly of MPH and antibodies on S-layer nanostructure for multifunctional applications. Adapted with permission from ref 161. Copyright 2015 John Wiley and Sons, Inc.

mosaic virus (TMV) have been built through genetic insertion or the chemically modified biotin−STV connection of GFP due to the inherent thermal stability of these viral capsids to maintain the specific interactions between individual CP subunits.166−168 This surface-display technology was also applied to attach other functional proteins on different viral carriers as long as sufficient CP-fusion proteins are available. A series of such AB-type conjugates (e.g., scFv−potato virus X (PVX),169 anthrax receptor 2 (ANTXR2)−Flock House virus (FHV),170 maltose binding protein (MBP)−FHV,171 and Borrelia burgdorferi outer surface protein (BbOsp)−hepatitis B virus (HBV))172 have been well-established based on whole virus particles or their split parts to develop morphologically distinct heterohybrid protein particles for future applications in environmental remediation and vaccine design. The same concept was applicable to the fabrication of extremely stable multiprotein complexes. For example, homododecameric disclike stable protein 1 (SP1) with resistance to diverse pHs, high temperatures (Tm of 107 °C), organic solvents, and various proteases was utilized as a novel 3D molecular scaffold for display GOx.173 The recombinant protein containing the GOx enzyme fused to the N-terminus of SP1 with a 23-aminoacid peptide linker to increase its structural flexibility was able to assemble into two different types of aggregates, namely, nanorings and nanotubes, which result from the self-assembly and further stacking of SP1 proteins, respectively (Figure 26). 13587


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of the recombinant proteins were precisely controlled by their length and hydrophilic/hydrophobic proportions to facilitate the self-assembly of biomimetic membrane structures.174 The synthetic constructs were based on two types of homopolymeric protein domains, namely, 20 hydrophilic VPGEG repeat units (E20) as well as 20 hydrophobic VPGFG repeat units (F20), and the hydrophilic ends were fused with monomeric enhanced GFP (mEGFP) domain for better characterization of their expression, distribution, and assembly using epifluorescence microscopy, TEM, internal reflection fluorescence (TIRF), and TIRF-structured illumination microscopy (SIM). As shown in Figure 27a, mEGFP−E20F20 was able to form

Figure 26. Self-assembly of GOx enzymes into nanorings and nanotubes on stable SP1 templates via genetic fusion. Adapted with permission from ref 173. Copyright 2007 American Chemical Society.

Owing to the proximity effect between two fused proteins, an immobilized enzyme monolayer on external surfaces of SP1 assemblies showed a higher inactivation time and a longer halflife than native GOx. Biological templates offer the programmed scaffolds to immobilize multiple proteins with precise spatial control through selective biochemical interactions or genetic fusion for the fabrication of high-density surface displays. This biomimetic strategy takes advantage of biomolecular superscaffolds that are sufficiently rigid and have the ability to organize vast amounts of topologically defined architectures for specific protein arrangements, from simple periodic arrays to complex patterns. It should be noted that the formation of protein superstructures into any deliberately designed architecture strongly relies on the recognition properties and structural features of the template molecules or motifs. A large number of components are required to dictate the precise positioning of protein molecules by mixing them in exact stoichiometric ratios for successful assembly. Thus, the high cost of biological synthesis and the high error rate of selfassembly are two major obstacles for biological-templateinduced protein self-assembly.

Figure 27. In vivo self-assembly of genetically encoded artificial proteins into organelle-like structures (a), 2D layer-like structures (b), and 3D hexagonal structures (c). Adapted with permission from refs 174, 175, and 176. Copyright 2015 Nature Publishing Group (ref 174) and 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim (refs 175 and 176).

2.5. In Vivo Protein Self-Assembly

well-defined organelle-like structures, and the translational order and ratio of E20/F20 domains have a significant influence on their self-assembly process in vivo. Furthermore, new biological functionalities such as the unnatural amino acid paraazido-L-phenylalanine (pAzF) can be site-selectively incorporated into the amphiphilic proteins for developing enzymatic membrane reactors with controllable catalysis. Several other studies have confirmed that the genetic fusion of distinct assembly factors could endow newly designed artificial proteins with the desired self-association properties in vivo. Multicomponent enzyme−protein complexes were constructed in E. coli BL21 (DE3) by coexpressing leucine dehydrogenase (LDH)−PDZ domain (LPd) and formate dehydrogenase (FDH)−PDZ ligand (FPl) genes from different sources on two separated plasmids, and subsequently performing scaffold-free coassembly into supramolecular networks for recycling catalysis (Figure 27b).175 The formation of highly ordered 2D layer-like structures relied on PDZ−ligand interactions as well as homologous oligomerization properties

Although in vitro self-assembly opens up new avenues to study and understand the dynamic behaviors of natural protein aggregates, the question of how to design proteins that spontaneously achieve the hierarchical organization processes in a complex cellular environment presents an imminent challenge. Inspired by the assembly/disassembly of endogenous protein monomers regulated by relevant enzymes and cofactors, it is possible to use several auxiliary factors to assist or direct an efficient protein assembly in vivo. These assembly factors can be incorporated into the protein leading to selfassembly or can self-assemble on their own, in which case the protein subsequently interacts with the initial scaffold, forming the final nanostructure. Synthetic biology has emerged as a powerful method for expression and in vivo assembly of artificial protein systems by forcing the cell to comply with the instructions encoded by de novo designed heterogeneous genes. Schiller and co-workers applied this technology to create novel amphiphilic proteins in living E. coli cells, in which the physicochemical characteristics 13588


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achieve a better monodispersity for DNA-like gel-based purification. Therefore, the discrete oligomeric states can be precisely controlled as versatile scaffolds for multivalent display of functional proteins such as MBP, mCherry, and protein G. Upon introduction of GFP11-capped mature GFP as a coexpression partner, the assembly process of GFP oligomers was significantly influenced by the blocking effect, resulting in the conversion of polygonal oligomers into linearly opened oligomers for more diverse protein display. As another example, GFP was engineered with a cationic peptide and a hexahistidine (H6) as N/C-terminal tags to direct the self-assembly of structurally diverse nanoparticles in CXCR4-expressing cells.178 These nanoparticles have a uniform spherical morphology and tunable sizes ranging from 15 to 30 nm (Figure 28b), which are strongly influenced by the ionic strength and amino acid composition of cationic peptides. Arginine-rich cationic peptides, including R9 (9-Arg peptide) and T22 (18 amino acids with positively charged 9-Arg/Lys), were shown to facilitate electrostatic protein−protein contacts in nanoparticle formation. The effect of these peptidic tags on renal clearance and biodistribution were monitored, and fluorescence accumulation in the target tissues rather than the kidney indicated the high in vivo architectural stability of R9-GFP-H6 and T22GFP-H6 nanoparticles. Because of the low demand for protein building blocks, this tag-based strategy could be applied to any protein for in vivo self-assembly in medical applications. An alternative route to design a self-assembling artificial protein is the engineering of chelating ligands onto monomeric protein surface for metal-directed in vivo assembly. Recently, a Zn2+-mediated supramolecular protein assembly was developed by Song and Tezcan within E. coli cells.179 The four-helixbundle haem protein, cytochrome cb562 (cyt cb562), was mutated into a variant (C96RIDC1) containing a T96C mutation, two bis-His motifs (H59/H63 and H73/H77), and six hydrophobic mutations (R34A/L38A/Q41W/K42S/ D66W/V69I) in different C2-symmetric interfaces and was subsequently self-assembled into a stable D2-symmetric tetramer (Zn4:C96RIDC14) held together by a combination of disulfide bridging, metal coordination, and interfacial hydrophobic interactions. As shown in Figure 29, the disulfide-crosslinked interfaces of Zn4:C96RIDC14 offered several appropriate positions for building four types of tetrahedral Zn2+−OH2/ OH− coordination motifs (AB1−4) as hydrolytic sites by the incorporation of three new residues (His and/or Glu) into C96 RIDC1 monomers, in which only the variant with AB3 exhibited very high efficiency (>90%) toward tetramerization upon Zn2+ binding. To confirm the C96RIDC1−AB3 tetramers in E. coli cells, an additional mutation (Lys104Ala) was required to eliminate the remarkable influence of the Lys104 side chain on ampicillin hydrolysis activity. As a result, both in vivo βlactamase activity and in vitro SEC analysis of the cellular extracts have clearly demonstrated the formation of the desired tetrameric states. In fact, more mutants could be constructed based on the tetrameric C96RIDC1-AB3 to achieve higher enzymatic activity for biomedical applications. In contrast with protein design and engineering, the utilization of in vivo nucleic acid-based nanostructures as scaffolds to arrange protein molecules could avoid some unexpected problems such as protein misfolding, insolubility, or inactivation.180 Silver and co-workers showed that synthetic RNA modules can be rationally programmed into 1D and 2D arrays for targeting proteins to specific locations on their

of octameric LDH and dimeric FDH. Fluorescence complementation analysis enabled direct visualization of these supramolecular structures in cells through the fluorescent signal triggered by the association of two parts of a red fluorescent protein (mCherry) that were fused to the Cterminus of LPd and the fusion joint of FPl, respectively. A successful example has also included the expression of a selfassembling fusion protein in living HeLa cells on the basis of the heavy chain of human ferritin (HuFtH) and the citrine fluorescent protein (CFP) (Figure 27c).176 A combination of the oligomerization abilities of two protein domains can result in the formation of 3D hexagonal structures, which were mediated by site-directed mutagenesis at hot spot position 206 to affect the stabilization of dimeric CFP interface. Using proper engineering designs, natural protein monomers can be reconstituted by the manipulation (e.g., insertion, deletion, and modification) of their genetic sequences for cellular protein assembly. Jung and co-workers reported the removal of the β-strand 11 fragment (GFP11, amino acids 215−230) from GFP and further fusion of this split segment to the N-terminus of the truncated GFP1−10 fragment (amino acids 1−214) by an optimized tripeptide linker in order to produce a recombinant nonfluorescent GFP with self-assembly behavior (Figure 28a).177 A mixture of GFP oligomers, varying

Figure 28. (a) Reengineered GFP monomer underwent self-assembly into polygonal structures or linearly opened oligomers for diverse protein display in E. coli BL21 cells. Adapted with permission from ref 177. Copyright 2015 Nature Publishing Group. (b) Spherical protein nanoparticles formed by different proteins through cationic peptideinduced self-assembly in CXCR4-expressing cells. Adapted with permission from ref 178. Copyright 2014 American Chemical Society.

from dimers to decamers, was accumulated in E. coli BL21 cells based on the irreversible, noncovalent association of two complementary GFP fragments, and the green fluorescence was recovered due to the formation of mature GFP. Highly charged GFP oligomers were subsequently prepared by systematic replacement of surface-exposed amino acids with Asp or Glu to 13589


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assemblies in vivo can be accomplished using the RNA inhibitory strands (ISs) to prevent their assembly processes. This was also demonstrated by a fluorescence complementation system (e.g., the split GFP fragments) that was attached to ferredoxin or hydrogenase to determine protein−protein associations based on the increased fluorescence.

3. CHEMICAL STRATEGIES FOR PROTEIN ASSEMBLY The second major class of protein assembly is featured by the induction of driving forces based on chemical strategies. On one hand, chemical strategies can reengineer natural supramolecular interactions including receptor−ligand interactions, metal coordination, and electrostatic interactions in a new fashion to dictate the interacting modules into ordered protein assemblies. On the other hand, chemical strategies can integrate natural or non-natural elements including receptor−ligand interactions, synthesized supramolecular interactions, and various templates to construct new protein assemblies. Therefore, chemical strategies have vastly expanded the scope of protein assembly by manipulating the structural elements and functional groups within the protein complexes. In this section, several main chemical strategies that guide protein assembly will be discussed.

Figure 29. Haem protein cyt cb562 with surface-engineered chelating ligands for metal-directed self-assembly in E. coli cells. Adapted with permission from ref 179. Copyright 2014 The American Association for the Advancement of Science.

3.1. Receptor−Ligand Interaction-Directed Protein Assembly

surface in BL21-star (DE3) cells.181 The RNA strands were designed to contain two functional domains, in which polymerization domains (PDs) folded back on themselves to form hairpin loops, and their stem structures together with dimerization domains (DDs) were tightly packed against each other by complementary base pairs to construct higher-ordered architectures through isothermal self-assembly (Figure 30). The resulting RNA scaffolds have retained the ability to insert multiple specific RNA aptamer domains (e.g., PP7 and MS2 aptamers) for selective recognition and capture of ferredoxin and hydrogenase, leading to spatially addressable multiprotein nanoarrays. Identification of these RNA-templated protein

Nature evolves with abundant receptors for stereospecific and reversible recognition of the particular ligands through noncovalent intermolecular interactions. Due to the innate diversity, specificity, and high affinity of receptor−ligand interactions, chemically operating the ligands to guide protein assembly is made possible by selective modification of the receptor proteins with surface-attached ligands or synthesis of various ligand-functionalized compounds to induce protein− protein associations and control the overall architectures of protein assemblies. Typical receptor−ligand interactions including streptavidin/avidin−biotin interactions, apoprotein− cofactor interactions, enzyme−inhibitor interactions, and lectin−sugar interactions have been utilized to enrich the toolbox for chemically directed protein assembly. 3.1.1. Heme−Hemeprotein Interaction Pair. Hemoproteins, such as hemoglobin, myoglobin, cytochromes, and P450 peroxidases, are a family of cofactor heme-containing proteins.182 As a typical receptor−ligand pair, the high affinity of apoprotein for hemes was explored to develop novel protein assemblies. Inspired by the principles of “bottom-up” nanobiotechnology, Hayashi and co-workers developed a series of protein self-assembly systems via supramolecular polymerization of hemeproteins based on the strong heme−heme protein interactions.183 As an example, they started with a fourhelix bundle hemoprotein, E. coli cytochrome b562 (cyt b562), the His63 residue of which was located on the opposite side of the heme-binding pocket and was mutated into cysteine to allow the linkage of a chemically synthesized heme cofactor derivative.184 After surface modification, the resulting protein variants underwent an acid denaturation and subsequent neutralization cycle to spontaneously give rise to 1D head-totail protein nanolines stabilized by heme−heme pocket interactions (Figure 31). SEC and AFM analysis confirmed that the formation of stable supramolecular protein polymers with different sizes was governed by the linker length, and their polymerization degree was calculated to reach 100 units. The dissociation of protein clusters that was affected by the

Figure 30. Spatially addressable multiprotein 1D and 2D arrays templated by aptamer-tagged RNA scaffolds in BL21-star (DE3) cells. Adapted with permission from ref 181. Copyright 2011 The American Association for the Advancement of Science. 13590


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Figure 31. Supramolecular hemoprotein polymerization induced by heme−hemeprotein interactions. Adapted with permission from ref 184. Copyright 2007 American Chemical Society.

Figure 32. (a) Affinity of lectin LecA for the tetragalactosylated 1,3alternate calix[4]arene-based multivalent glycocluster. (b) AFM images of LecA filaments. (c) Simulated model of a LecA filament. Adapted with permission from ref 191. Copyright 2011 Royal Society of Chemistry.

competition binding of native heme revealed that the protein polymerization was driven by reversible heme−apoprotein interaction. Morerover, heme−hemeprotein interaction pairs can also be used to create extended structures, such as 2D branched networks185 and 3D functional protein networks,186 through chemical diversification, which has been described in a previous review.187 3.1.2. Lectin−Sugar Interaction Pair. Another important receptor−ligand pair is sugar and its binding protein, lectin. Sugar−lectin interactions have been found to play key roles in numerous biological processes including fertilization, adhesion and virulence of pathogens, and inflammatory response.188 Lectin concanavalin A (Con A) is a typical member of the lectin class. It consists of four subunits, each of which features two antiparallel pleated sheets and a binding site on the outer surface to recognize saccharides such as α-D-mannose and α-Dglucose.189 Inspired by earlier work on the design of 3D protein crystals through interactions between tetrahedral Con A and α-Dmannopyranoside,190 a multivalent glycocluster was developed to provide better control of the lectin protein self-assembly.191 Vidal and co-workers synthesized a tetragalactosylated 1,3alternate calix[4]arene-based glycocluster with nanomolar affinities for LecA by taking advantage of the multivalency and proper topology of the sugar moieties (Figure 32a). AFM images showed that highly organized 1D lectin filaments were formed and interrupted by bifurcations and branching points (Figure 32b). The branches in linear regions can be justified by the defect in the symmetry of the glycocluster because one of the four sugar moieties can bind to a third lectin on the side of the filaments to generate a branching point (Figure 32c), thus revealing the capabilities of multivalent lectin−sugar interactions to control the morphology of protein assemblies. 3.1.3. (Strept)avidin−Biotin Interaction Pair. The (strept)avidin−biotin interaction is one of the strongest noncovalent interactions in Nature, with an association constant reaching up to ∼1015 M−1.192 Normally, noncovalent host−guest binding affinities in water are typically in the range of 101−106 M−1, and protein−ligand binding rarely exceeds 1011 M−1.193 (Strept)avidin is a tetramer, and each subunit contains eight β-strands that form an antiparallel β-barrel-

shaped structure. A hydrophobic pocket is “buried” in the core of each β-barrel that facilitates the binding of biotin with high affinity and selectivity. This receptor−ligand pair has been widely utilized in chemical and biological applications.194,195 In fact, it also offers an important driving force for the construction of highly ordered protein assemblies. Ringler and Schulz reported the first example of protein assembly by avidin−biotin interactions.196 A C4-symmetric tetrameric L-rhamnulose-1-phosphate aldolase (RhuA) with a His6-tag fused to its C-terminus was chosen as the four-arm junction. Multiple site-directed mutageneses were performed to remove the interfering thiol and introduce two cysteines into each subunit for appending a total of eight biotins on the side surface of aldolase via a thiol−disulfide exchange reaction. The resulting aldolase tetramer exhibited specific interactions with D2-tetrameric STV due to the extremely high affinity between STV and biotin. Therefore, a series of complex patterns can be constructed by mixing STV and bisbiotin-labeled STV molecules as building blocks (Figure 33a). Notably, the length of the tethered biotin should be short enough to ensure the specific binding to the side surface of streptavidin. Moreover, a dynamic protein network with a switchable mesh can be achieved by utilizing conformational responsive proteins (e.g., a Ca2+-binding β-helix fragment of the enzyme serralysin from Serratia marcescens) as spacers within the network (Figure 33b). In this study only limited sizes of protein arrays were observed, probably owing to the flexibility of the linker and imperfect control over the assembly orientation. 3.1.4. Other Interaction Pairs. Apart from typical receptor−ligand interactions mentioned earlier, other interactions, such as enzyme−inhibitor and peptide−protein interactions, have also been selected to guide protein assembly. Considering that most receptor−ligand interactions are nondirectional and relatively flexible, researchers have paid much attention to the construction of protein nanorings via a polymeric ring−chain competition mechanism.197 As a typical example, Meijer and co-workers prepared an AB monomer with self-associating S-protein (A end) and S-peptide (B end) of the ribonuclease (RNase).198 To quantitatively analyze the ring− chain competition mechanism during the thermodynamically 13591


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product distribution with the theoretical values using the ring− chain competition model. The AB monomer with longer EG chains (AB1) shows great agreement with the Jacobson− Stockmayer theory,199 while AB2 with relatively shorter EG connectors shows large deviations with calculated values at high concentrations, which may be attributed to the inherent strain in the cyclic aggregates formed by short chains. This work elegantly demonstrated the great influence of the length of the linker and concentration of the building block on the final protein self-assembly architecture, which established guidelines toward the design of momoneric precursors to protein-based supramolecular polymer. Following the same ring−chain competition mechanism, Wagner and co-workers synthesized 2D enzyme nanorings by the interactions of fusion protein dimer (E. coli dihydrofolate reductase molecules that were tethered together by a flexible peptide linker ecDHFR2) and dimeric enzyme inhibitor, bisMTX-C9 (Figure 35).200 TEM images provided a direct

Figure 33. (a) Rational design of biotin-labeled tetrameric aldolase (bR) to create a noncovalent planar network or rods by binding STV (S) to bisbiotin-labeled STV spacer (bbS); (b) PGAL-β-PGAL fusion protein designed for future fabrication of a Ca2+-responsive network. Adapted with permission from ref 196. Copyright 2003 American Association for the Advancement of Science.

controlled self-association process, both S-proteins and Speptides were connected via a flexible oligo(ethylene glycol) (EG) linker. Two AB monomers contained flexible EG chains of various lengths (AB1 and AB2) that could reversibly assemble to form supramolecular protein complexes with enzymatic activities, which demonstrated that the affinity between selfassociating parts is sufficient to dictate protein self-assembly. Further SEC and quadrupole time-of-flight (Q-TOF) mass analysis validated the formation of cyclic protein assemblies (Figure 34). Consequently, the authors also compared the

Figure 35. Schematic representation of the bis-MTX and DHFR2 system self-assembling into protein nanorings via the ring−chain competition mechanism. Adapted with permission from ref 200. Copyright 2006 American Chemical Society.

observation of the toroid protein oligomer formation. Further experimental data together with theoretical analysis confirmed that the size distribution of the nanorings can be tuned by adjusting the interdomain linker length, revealing that the formation of nanorings is governed by the subtle balance between entropy and conformational dynamics. Notably, the protein nanorings exhibited size-dependent catalytic efficiency, which offers a possible methodology to regulate catalytic parameters via conformational control.201 Through fusing an anti-CD3 single-chain variable region to the building block protein (DHFR), the previous DHFR nanorings can further be utilized as templates to reversibly control the assembly or disassembly of the mimetic antibodies.202 The anti-CD3 chemical induced antibody nanorings maintain a high affinity and exhibit similar cellular internalization mechanism.203 Furthermore, a proof-of-concept work demonstrated that the DHFR2anti-CD3 proteins can be used for the targeted cellular delivery of various cargos including oligonucleotides, conjugated small molecules, and proteins via the labeling of oligonucleotides to the bis-MIX (for details, see section 4.3).204 Apart from utilizing single proteins or their oligomers as building blocks, Belcher and co-workers utilized a large

Figure 34. Schematic representation of the ring−chain supramolecular polymerization of RNase S building blocks. Adapted with permission from ref 198. Copyright 2010 Royal Society of Chemistry. 13592


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filamentous virus complex as a building block to construct twodimensional nanorings.205 One end of the filamentous M13 virus was engineered with a His6-tag as pIX fusion, and the other end was engineered with antistreptavidin peptide as pIII fusion to yield the bifunctional virus. A heterobifunctional linker molecule of streptavidin−Ni-NTA (NTA = nitrilotriacetic acid) was chemically synthesized by an 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC)-catalyzed reaction between primary amine groups of NTA and carboxylate groups on streptavidin. As demonstrated by AFM characterization, at a low concentration and incubation with one stoichiometry of the linker molecule, the filamentous virus can bend into protein nanorings under the cooperation of biotin/streptavidin interactions and metal coordination (Figure 36). The M13

One example in this regard is the combination of heme− hemoprotein and avidin−biotin interactions in the 2010s. Hayashi and co-workers designed a dyad small molecule bearing a heme moiety for hemoprotein binding and a bisbiotin unit for the exclusive association with streptavidin.206 Onedimensional alternating protein coassembly was obtained via utilizing a disulfide-bond-linked myoglobin mutant dimer (MbA125C)2 and streptavidin as building blocks (Figure 37).

Figure 37. Schematic representation of the design of an alternating protein copolymer by combining heme−hemoprotein and avidin− biotin interactions. Adapted with permission from ref 206. Copyright 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 36. Schematic representation of the nanorings formation of the bifunctional M13 viruses with antistreptavidin peptide and hexahistidine peptide, respectively, at opposite ends. Adapted with permission from ref 205. Copyright 2004 American Chemical Society.

SEC analysis confirmed the formation of protein copolymers with large molecular weights under thermodynamic control, which was dependent on the length of the linker as well as the ratio and the concentration of the building block. In addition, AFM images of washed and dried samples revealed that the periodicity of the aligned spherical dots is consistent with the theoretical value, which further confirmed the formation of a heterotropic protein copolymer. Another important development of cooperative protein selfassembly by integrating different noncovalent interactions is the combination of receptor−ligand interactions with metalcoordination interactions. Ward and co-workers described a hierarchical protein self-assembly system utilizing this kind of cooperation.207 Similarly, the design also began with the synthesis of the divalent linker (Biot2-terpy) bearing a terpyridine and two biotin moieties. The Biot2-terpy linkers were able to coassemble with the [Fe(Biot2-terpy)2]2+ complex under the coordination of ferrous ions in a cooperative fashion (K1 = 1.26 × 107 M−1, K2 = 6.31 × 1013 M−1). The subsequent addition of streptavidin into the [Fe(Biot2-terpy)2]2+ complex exclusively afforded one-dimensional protein assemblies (Figure 38), which could be monitored by the disappearance

virus nanoring retained the binding affinity with polyclonal pVIII primary antibody and subsequent recognition of the second antibody (antirabbit IgG) that can conjugate 10 nm gold nanoparticles, which shows great potential for guiding nanoparticles assembly through these constructed protein structures as templates. 3.1.5. Cooperative Receptor−Ligand Interactions. In Nature, most protein self-assemblies usually involve the cooperation of multiple supramolecular interactions. The integration of receptor−ligand and other supramolecular interactions in a single self-assembling system offers unique advantages to direct protein self-assembly, in which heteroligands can be synthesized for protein−protein associations and the protein building blocks do not require any chemical or biological modification to achieve highly selective and directional assembly processes. This novel design strategy represents a convenient way to construct complicated protein nanostructures through the chemical integration of orthogonal supramolecular interactions. 13593


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Figure 38. Schematic representation of the concept of streptavidin self-assembly dictated by the cooperation of metal-coordination and receptor−ligand interactions. Adapted with permission from ref 207. Copyright 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim. Figure 39. (a) Chemical structures and schematic illustrations of the competitive binding between biotinylated and desthiobiotinylated DNA strands toward biotin. (b) Illustration of the nonsensical Morse code and corresponding AFM images. (c) Revelation of the encrypted Morse code message “NANO” with the addition of biotin and corresponding AFM images. Adapted with permission from ref 208. Copyright 2013 American Chemical Society.

of an induced CD signal at 505 nm. Scanning electron microscopy (SEM) experiments indicated that the induced protein polymers can subsequently undergo hierarchical selfassembly to form millimeter protein bundles under the chelation of calcium between lateral streptavidin surfaces. Significantly, the resulting protein bundles can act as templates for the biological mineralization of calcite microcrystals in the presence of a CO2 source and CaCl2 solutions. In an alternative study, selective reversible receptor−ligand interactions can be elegantly utilized to encode a nanoscale Morse code message onto DNA-origami templates. Specifically, Lu and co-workers prepared an origami tile consisting of 9 desthiobiotinylated and 15 biotinylated staple strands (Figure 39a), and the word “NANO” was encoded within the arrangement of the strands.208 When the tile was incubated with streptavidin, the tile was completely covered with proteins and the encrypted message was not visible (Figure 39b). However, by taking advantage of the different binding affinities of biotin and desthiobiotin toward streptavidin, upon subsequent addition of the “decoder” (excess biotin), the original intended message “NANO” was revealed (Figure 39c). To further examine the reversibility of this design protocol, it was demonstrated that the letter “I” could be replaced with “i” and subsequently restored back to its original form “I”. This design strategy bodes great potential within the fields of nanoeletronics, photonics, and biomedicine where programmable capture and release of nanomaterials are desirable. More accurate protein assemblies can be achieved by employing synergistic multiple driving forces. Song and Jiang and their co-workers adopted a dual strategy to construct 3D protein crystals by combining lectin−sugar interactions and hydrophobic ligand dimerization.209 The designed heteroligand consists of a rhodamine B (RhB) moiety and a sugar moiety that are tethered by oligo(ethylene oxide). The cooperation between RhB dimerization and lectin−sugar interactions contributes to the formation of 3D lectin ConA crystals, and their steric structures can be further regulated by adjusting the length of heteroligand linker as characterized by X-ray crystallography (Figure 40a, b). A kinetics study suggested that the crystallization mechanism proceeded via a two-stage

Figure 40. 3D protein arrays induced by dual supramolecular interactions of protein−sugar interactions and π−π stacking. (a) Protein crystals. (b) Layer structures regulated by the length of the heteroligand linker. Adapted with permission from ref 209. Copyright 2014 Nature Publishing Group. (c) Helical protein microtube. Adapted with permission from ref 210. Copyright 2016 American Chemical Society.

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self-assembly process: first, the lectin became immediately bound to the sugar moiety, and the dimerization of the RhB moiety was subsequently accomplished to create the final crystalline structure. The unique sequential binding kinetics of this system allows for a rapid and high-yield crystallization under excess ligand conditions when the building blocks are not end-capped. This strategy has also proven to be successful in mimicking protein microtubules with precise structures and controllable self-assembly behavior by the same group.210 Dual supramolecular interactions of protein−sugar interactions and π−π stacking were introduced into a new tetrameric protein system, soybean agglutinin (SBA), to construct highly homogeneous protein microtubes. Cryo-TEM characterization and computational modeling of the left-handed helical microtubular structures showed that three protofilaments build into the microtube wall by twisting around a hollow core and each of them has a periodic helical structure with nine SBA tetramers that are linked by the well-designed ligands (Figure 40c). Most strikingly, both structural and dynamic features have been reproduced in the artificial protein microtubule as compared to the natural ones. The enhanced immune response to macrophage cells could also endow the protein microtubes with immunological functions for biological applications. As described above, naturally occurring receptor−ligand pairs with their own advantages of high affinity, selectivity, and reversibility have been successfully used to direct protein assembly. This strategy can combine the accuracy and reliability of interprotein interactions and the diversity of chemical synthesis to produce homo- or heteromultivalent ligands for promoting the construction of protein assemblies. Because most examples do not require any chemical modifications of the protein surfaces, the resulting assemblies can retain the protein’s integrity and bioactivity for various functions such as catalysis and sensing. The further development will focus on the combination of multiple driving forces, the construction of more complicated protein assemblies with various functions, and the realization of biological and material applications. Furthermore, particular emphasis will be devoted toward exploiting new receptor−ligand pairs for protein assembly.

Combining all of these advantages, scientists have utilized metal coordination to induce a wide variety of self-assembled protein superstructures. Tezcan and co-workers pioneered the concept of treating proteins as large ligands and employing them to directly dictate protein self-assembly via metal coordination.214 Their early work focused on the demonstration of the accessibility of this strategy via fabrication of a series of protein oligomers. To start with, a relatively simple four-helix bundle cytochrome cb562 (cyt cb562) was selected as a model building block. The C2-symmetrical interface that was characterized by the crystallography of natural cb562 provided the starting point, on which two bis-His motifs (His59/His63 and His73/His77) were incorporated to generate the variant (MBPC-1) for selectively binding with metal ions. Sedimentation velocity (SV) and crystallography analysis confirmed metal coordination to be a powerful tool to dictate protein self-assembly, in which a tetrameric assembly with V-shaped MBPC-1 pairs wedged into one another were observed to be stabilized by Zn(II) ions (Figure 41a).215

Figure 41. (a) Crystal structure of a 4 Zn/4 His4-cb562 assembly. View of the assembly parallel to the noncrystallographic 2-fold axis and the corresponding cylindrical representation. (b) Close-up view of the Zn coordination environment. Adapted with permission from ref 215. Copyright 2007 American Chemical Society.

3.2. Metal-Coordination-Driven Protein Assembly

Metal ions play essential roles in biological systems, and nearly one-third of proteins are metalloproteins where metals are vital to their functions.211 Metal ions are able to act as cofactors and regulate enzyme-catalyzed pathways; the strong binding strength that can be achieved through metal coordination can stabilize quaternary protein superstructures; and the supramolecular nature of metal coordination allows for the regulation of transient protein−protein interactions during dynamic cellular processes. Nature has demonstrated that metal coordination provides an excellent tool to induce protein selfassembly. Amino acid residues including histidine (His), cysteine (Cys), aspartic acid (Asp), and glutamic acid (Glu) can act as ligands to donate electron density to the metal ions, among which the imidazole substituents in His residues are the most prominent donors.212 Thus, a bis-His clamp is a commonly designed metal-chelating site for selective metal binding among all the ligands on heterogeneous protein surfaces.213 Considering the fact that His is a natural amino acid residue, it is feasible to modify protein surfaces via genetic mutation.

Further analysis revealed that the overall geometry of the protein self-assembly can be controlled by the innate stereochemical preferences of the metal ions. Crystal structures of Cu(II) and Ni(II) complexes of MBPC-1 exhibited distinct C2-symmetric dimer (Cu2:MBPC-12) and parallel C3-symmetric trimer (Ni2:MBPC-13) structures, respectively.216 This finding demonstrates that the directionality of the metal coordination can dominate the supramolecular arrangement of protein selfassemblies. However, notably, the detailed and subtle interaction modes within the metal-dictated protein assemblies are the comprehensive interplay of the metal-coordination and noncovalent protein−protein interactions under thermodynamic control. For example, the former Zn4:MBPC-14 complex is coordinated by an Asp74 residue located within the 73/77 bis-His clamp instead of the originally designed His59 residues (Figure 41b). To illustrate this slight deviation, MBPC-2, the D74A/R62D variant of MBPC-1, was engineered and 13595


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complexed with Zn ions. The crystal structure of Zn4:MBPC-24 exhibited a D2-symmetrical V-shaped architecture, within which each Zn ion was chelated by the 73/77 bis-His motif from one monomer, His59 from a second, and His63 from a third. The similarity between Zn4:MBPC-14 and Zn4:MBPC-24 suggested that tetrahedral Zn coordination dictated the D2 symmetry of the assembly, whereas the distinct salt bridge formed within each crystal altered the noncovalent protein-interface interactions (Figure 42), thus subsequently influencing the energy landscape of the self-assembly process and ultimately giving access to different oligomeric conformations.217

Figure 43. Zn-mediated RIDC3 self-assembly under fast and slow nucleation conditions. Adapted with permission from ref 218. Copyright 2012 Nature Publishing Group.

line arrays even under fast nucleation conditions, which suggests that the self-assembly pathway can be regulated by tuning the intermolecular interactions. In contrast to RIDC3 monomers, Zn-mediated nanotubes and 2D arrays show significant improvement in stability against high temperature and organic solvents. This could facilitate the functionalization of the assembly when the distinct thermal stability of the protein complex was emphasized and exploited for the preparation of monodispersed Pt nanoparticles based on the stable protein nanoscaffolds. More interestingly, dynamic optical-stimuli responsiveness can be easily tailored into the nanoparticle fabrication process by replacement of the iron heme of protein lattices with photosensitive zinc heme (Figure 44).219 In comparison with the work of Tezcan and co-workers that mainly focused on the construction of protein self-assembly crystals and exploration of their kinetic/thermodynamic mechanism, Liu and co-workers paid more attention to the manipulation of protein self-assembly behavior in solution at relatively low protein concentrations. A globular dimeric enzyme-glutathione transferase from Schistosoma japonicum (sjGST) was chosen as the building block for metal-dictated protein self-assembly. His 6-tag, a traditional ligand for Ni2+coordination, can be genetically fused to various proteins to improve the protein-purification procedures and is also an ideal candidate for driving the metal-directed linear supramolecular self-assembly of natural enzymes. To prove the possibility of utilizing metal coordination to direct sjGST selfassembly, a GST mutant with a His6-tag attached to the Nterminus (sjGST-6His) was designed. The high-performance liquid chromatography (HPLC) measurements along with the AFM characterizations validated that fascinating protein nanowires were successfully constructed by chelating with Ni2+ ions (Figure 45a).220 Considering that GST is a natural

Figure 42. Distinct interfacial hydrogen-bonding interactions in (a) Zn4:MBPC-14 (hydrogen bonds between R34 and D66) and (b) Zn4:MBPC-24 (hydrogen bonds between R34 and D62). Adapted with permission from ref 217. Copyright 2008 American Chemical Society.

On the basis of the foundations laid by the oligomer studies, the same group extended their investigations to a realm of extended 1D, 2D, and 3D molecular arrays. Derived from the structure of Zn4:MBPC-14, a new mutant (RIDC3) featuring 10 incorporated Rossetta design surface mutations was constructed for the stabilization of a C2-symmetrical dimer (Zn2:RIDC32). The open coordination sites for binding to another dimer allow the formation of extended protein superstructures. TEM analysis revealed that RIDC3 can kinetically assemble into 1D nanotubes under fast nucleation or stack into thermodynamically stable 2D and 3D planar arrays under slow nucleation, which can be predictably tuned by external stimuli such as metal concentration and pH (Figure 43).218 In-depth X-ray analysis revealed that 3D Zn-RIDC3 arrays feature multiple metal chelating sites, and these strong Zn-mediated interactions are complemented by surface patches of polar interactions between the 2D layers. Real-space reconstruction suggested that both nanotubes and 3D arrays exhibit the same RIDC3 arrangement, which corroborates the interconversion process between the nanotubes and the sheetlike arrays. Intriguingly, rhodamine-modified RIDC3 variants with enhanced interlayer interactions were demonstrated to assemble into thermodynamically preferred crystal13596


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protein self-assembly processes. Liu and co-workers proposed a facile design strategy for the accurate control of sjGST selfassembling system with rational steric design and the cooperation of metal-coordination and protein−protein interactions.221 Two symmetric metal-binding motifs that vertically align with the C2 axis of sjGST dimers were designed and engineered onto the surface of sjGST in an opposite, “V”shaped orientation. The steric design provides the preference for assembling sjGST proteins into nanorings; however, the formation of the “V”-shaped structures alone cannot guarantee the final nanoring formation. Supramolecular control over the protein self-assembly process is essential: through computational calculations, all of the noncovalent protein−protein interactions between 2His-sjGST were studied, and in one specific docking model our designed metal-coordination force and interfacial nonspecific interactions were found to cooperate to determine the final interaction mode of the 2His-sjGST. Extending this interaction mode will give rise to ordered protein nanorings (Figure 45b), and AFM characterizations have validated this mechanism. Furthermore, the diameters of the protein nanorings have been further regulated by tuning protein−protein interface interactions (alteringing the ion strength). Although the construction of protein self-assemblies has gained preliminary success, the accurate control and predictable design of the final structure have been rarely reported. This study provides a facile entry for the construction of highly ordered protein nanorings and may inspire further construction of more complex protein superstructures. Another representative work was accomplished by Aida and co-workers, who aimed to construct a switchable, dynamic protein self-assembly system utilizing metal coordination as the driving force. Instead of mutated His-containing chelating sites, they utilized the photochromic units [spiropyran (SP)/ merocyanine (MC)] as the metal-chelating motifs and sitespecifically attached them onto the apical domains of the cylindrical chaperonin GroEL protein.222 Divalent metal ions are able to coordinate with MC to afford 1:2 complexes; thereby modified GroELs can form into long cylindrical nanofibers in the presence of divalent metal ions such as Mg2+, Ca2+, Mn2+, Co2+, and Zn2+. The resulting hollow cylinders exhibited high mechanical stability and maintained the capability of natural chaperon to bind with denatured proteins, thus serving as a novel biocontainer for delivering a variety of guest molecules. Light-induced SP/MC isomerization endows this system with a highly dynamic self-assembly characteristic, in which the switchable structure of photochromic moieties via a ring-opening reaction results in a controlled metal−ion complexation to mediate the assembly and disassembly processes (Figure 46).223 Upon exposure to UV light irradiation, nonionic SP is converted to ionic MC that can chelate with metal ions to direct the formation of GroEL nanotubes. Conversely, when exposed to visible light, SP is recovered and loses the ability to bind metal ions, leading to the scission of nanotubes into short segments. Adenosine-5′-triphosphate (ATP) provides an alternative manipulation for GroEL nanotubes due to the drastic conformational rearrangements of chaperonin protein during ATP hydrolysis, which produces a mechanical force to drive the disassembly of the nanotubes and thereby allows the trapped guest proteins to be released from its cavity after refolding.224 This process was also realized in a real cellular environment when boronic acid derivative modified nanotubes (BANT) were uptaken into HeLa cells for the investigation of ATP-responsive

Figure 44. (a) Photocatalytic cycle for the reduction of Pt2+ to Pt0. (b) Photoredox-mediated growth of PtNPs on ZnP-RIDC3 arrays. (c) Nonirradiated ZnP-RIDC3 arrays. (d) Irradiated arrays display uniform coverage with PtNPs that have a narrower size distribution. Adapted with permission from ref 219. Copyright 2014 National Academy of Sciences.

Figure 45. (a) Ni2+-induced linear assembly of sjGST-6His. Adapted with permission from ref 220. Copyright 2012 Royal Society of Chemistry. (b) Ni2+-induced assembly of 2His-sjGST into 2D nanorings. Adapted with permission from ref 221. Copyright 2013 American Chemical Society.

enzyme, the catalytic capacity of the metal-directed GST nanowires was evaluated and revealed that the enzymatic behavior of the native GST was well-conserved. As described above, the construction and preliminary functionalization of 1D protein self-assembly has been realized. However, moving forward, the next goal is to construct more complex architectures and achieve more precise control over 13597


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necessary to produce protein assemblies with a long-range order. 3.3. Electrostatic-Interaction-Induced Protein Assembly

Electrostatic interactions are key PPI regulators for proteins, where the attractive contacts between oppositely charged amino acids (e.g., Lys and Glu) make a significant contribution to the specificity of protein recognition, the stability of protein structures, and the formation of natural protein complexes.227 The utilization of these charges as a main driving force or manipulation of them to change the local dielectric properties of proteins for controlled protein assembly meets two challenges: (i) Electrostatic interactions are long-range cooperative ion effects that are unable to be accurately quantified. (ii) It is difficult to control the complex charge distribution on heterogeneous protein surfaces to form a relatively uniformly charged zone, and it is also varied with the pH value and ionic strength.228 In the practical design of large protein assemblies, one often needs to consider the careful selection of a proper protein unit and the combination of the steric geometric design and intrinsic charge characteristics of protein building blocks. Kostiainen et al. pioneered the concept of utilizing electrostatic interactions to guide the self-assembly of highly ordered patchy protein cages, including cowpea chlorotic mottle viruses (CCMVs) and ferritins.229−231 The negatively charged surface and structural symmetry determine that the building block can be readily assembled via electrostatic attractions. In a typical work,229 cationic dendrons featuring polyamine surface have been proven to serve as a powerful inducer to dictate CCMV self-assembly into densely packed aggregates (Figure 47a). As evidenced by DLS and electrophoresis measurements, in the presence of cationic dendrons, negatively charged CCMVs can undergo hierarchical assembly into secondary protein complexes, which is governed by the generation and concentration of the dendrons, as well as the ion strength of the solvent media. The dense hexagonal packing morphology of the complex is also clearly visible by TEM images (Figure 47b). Moreover, the system can be further functionalized with dynamic, optical responsiveness, which can be achieved by adding a designed o-nitrobenzyl linker between the positively charged polyamine surface and the dendritic core: when it is irradiated by long-wavelength ultraviolet light, the triggered photocleavage of the linker will subsequently release the original cationic surface and create negatively charged

Figure 46. Mechanism of the assembly/disassembly of spiropyran (SP)/merocyanine (MC)-labeled GroEL protein nanofibers controlled by UV/vis light as an external stimuli. Adapted with permission from ref 223. Copyright 2013 American Chemical Society.

intracellular drug delivery. In the biodistribution test, the BANT level in tumor tissues was much higher than that of healthy tissues, which shows great potential application in tumorspecific treatments. Coordination chemistry has made considerable progress in supramolecular construction of synthesized building blocks.225,226 The efforts in supramolecular coordination chemistry inspire scientists to develop metal-coordination strategies to control the self-assembly of biomolecules, and using the above-discussed strategies, a series of protein nanoarchitectures, some with special biological functions, have been created. In contrast to other diving forces, metalcoordination geometry, strength, directionality, and stereochemistry are the most prominent advantages when used to drive assembly strategy. These advantages allow the fine-tuning of the final structure of protein assemblies with less surface modification. Nevertheless, this strategy requires carefully controlled conditions (e.g., pH and metal concentration) to prevent the unexpected ligands involved in metal coordination that lead to the formation of undesired assembly structures. Such environmental sensitivity also endows metal coordination with the reversible and dynamic properties for self-healing

Figure 47. (a) Electrostatic-interaction-induced assembly and optically controlled disassembly of the protein−dendron complex. (b) Corresponding TEM images of the processes in (a). Adapted with permission from ref 229. Copyright 2010 Nature Publishing Group. 13598


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carboxylate functions, thereby dismantling the attractive electrostatic interactions and leading to the disassembly of the protein aggregates. Notably, the approach to construct and regulate electrostatic-interaction-based protein−dendron complex was also applicable for other negatively charged protein particles such as ferritin cages, revealing the generality of this strategy. Besides optical control, the electrostatic-interaction-induced virus−polymer complex can also be regulated by adjusting the temperature. Kostiainen et al. further utilized a dual-functional diblock copolymer consisting of poly(diethylene glycol methyl ether methacrylate) (poly(DEGMA)) and poly((2dimethylamino)ethyl methacrylate) (poly(DMAEMA)) as a cationic inducer. The poly(DMAEMA) block carries positive charges while the poly(DEGMA) block acted as a thermoresponsive switch: When the temperature is increased above the cloud-point temperature (Tcp) of the DEGMA block, the polymer chains collapse on the surface of the virus, which makes them partially hydrophobic, and they consequently aggregate into large hierarchical assemblies. When the solution temperature is reduced below the Tcp, the poly(DEGMA) blocks become rehydrated and free viruses can be released (Figure 48).230 This assembly/disassembly process is fully

Figure 49. (a) Building blocks of binary protein cage−nanoparticle superlattices. (b) (CCMV−AuNP8)fcc superlattices viewed along [110] and [111] projection axes and corresponding schematic frames. Adapted with permission from ref 232. Copyright 2012 Nature Publishing Group.

suggested this distinct superlattice configuration to be the comprehensive interplay of interparticle electrostatic interactions and steric fitting. Similarly, binary nanoparticle superlattices consisting of ferritins and AuNPs also can be prepared according to the same design concept. Normally, electrostatic interactions are considered to be undirectional and cannot be used to direct the self-assembly of heterogeneously charged protein particles into 3D crystal structures. Significantly, through subtle manipulation and delicate design, this work breaks the confinements and extends the scope of electrostaticintroduced protein self-assembly into three dimensions for the first time. As described above, the formation of hybrid superlattice structures that partly rely on synthetic particles has been successful. However, the integration of different natural protein-based building blocks into a single superlattice has remained elusive. To address this challenge, Kostiainen et al. proposed a new strategy where patchy cowpea chlorotic mottle virus (CCMV) particles (isoelectric point or pI ≈ 3.8) and avidin (pI ≈ 10.5) with oppositely charged net surfaces were assembled into a binary superlattice.233 SAXS and cryo-TEM studies verified the structure of the CCMV−avidin crystals as finite body-centered cubic (bcc) Bravais lattice structure. This distinctive bcc structure of CCMV−avidin crystals was found to be dictated by the distribution geometry of the patchy building block but not by the size ratio of the two components. Intriguingly, the relatively open CCMV−avidin superlattices allow further pre- or postfunctionalization with uncharged biotin-tagged functional units attributing to the innate biofunctionality of avidin (Figure 50). For example, the superlattice can be fluorescently labeled with biotin-conjugated dye molecules to allow for direct fluorescence measurements. Moreover, enzyme-active superlattices can be obtained via the incorporation of biotin-ligated HRPs; notably, the reaction velocity in the case of functionalized superlattice is ∼2 orders of magnitude higher in comparison with residue-free enzymes due to the presence of concentrated active sites loaded by periodic

Figure 48. Schematic representation of the possible assembly pathways between the poly(DEGMA-b -DMAEMA) copolymers and CCMV. Adapted with permission from ref 230. Copyright 2011 WileyVCH Verlag GmbH& Co. KGaA, Weinheim.

reversible and was shown to sustain several heating−cooling cycles. Moreover, the concept of electrostatic-interactioninduced CCMV self-assembly was demonstrated to not be limited to this type of dendron but is generally applicable to a wide range of cationic polymers.231 Moving forward, Kostiainen et al. made a significant advance from inducing the formation of a hierarchical densely packed complex to the construction of highly ordered protein superlattices with 3D periodicity. Spherical 1-pentanethiolstabilized AuNPs were utilized (Figure 49a) as cationic inducer instead of branching dendrons.232 As studied by SAXS and cryo-TEM measurements, when the electrostatic interactions are modulated to an optimized intermediate regime via regulating the Debye screening length and the pH value, highly ordered superlattices of CCMV/AuNP can be obtained. SAXS data and cryogenic electron tomography (cryo-ET) revealed that the CCMV/AuNP superlattice adopted a (CCMV−AuNP8)fcc superlattice structure (Figure 49b), which is unique and unprecedented. Deeper analysis of the cryo-TEM tomography and the crystal structure of CCMV 13599


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centered cubic (fcc) packed cocrystals retained the innate optical properties of Pc including fluorescence at 695 nm and efficient light-induced 1O2 production. This hierarchical selfassembly system combining functional moieties and patchy virus cages demonstrates a powerful methodology for the spontaneous creation of functional biohybrids, which should greatly advance the development of biomedicine and nanomaterials. The cagelike ferritins have been proven to be capable of undergoing assembly into dense aggregates or 3D superlattices. However, with delicate regulation, they can also assemble into 1D linear assemblies utilizing the channels on their surfaces. Zhao and co-workers utilized a reconstructed mature soybean seed ferritin (rmSSF) with a larger channel pore size of 1.2 nm in length and 0.4 nm in width as a building block.235 rmSSF’s can be induced by cationic poly(α,L-lysine) (PLL15 with a length of 4−5 nm) into a linear species through the 4-fold channel-directed electrostatic attraction at pH 7.0 (Figure 52). This work managed to modify the channels on protein surfaces and focus on the electrostatic interactions that were localized on the pores rather than the whole surface, which represents an alternative way to build electrostatic-interaction-induced protein self-assembly systems. Apart from patchy protein cages, Liu and co-workers utilized a double-layered six-membered ringlike stable protein one (SP1) dodecamer as the targeted building block. According to the crystal structure, SP1 protein features negatively charged top and bottom surfaces at neutral pH, combining with its highly ordered structure, suggesting that it can be employed as an appealing “brick” for electrostatic self-assembly. Positively charged quantum dots (QDs) with different sizes were synthesized to mediate the self-assembly of the protein rings into distinct morphologies, such as nanowires, subsequent bundles, and irregular networks in aqueous solution. As a proofof-concept attempt to construct functional protein-based biomaterials, the innate optical properties of the ideally arranged QDs within the assemblies were utilized to fabricate light-harvesting antennas. Fluorescence spectra indicated an obvious fluorescence resonance energy transfer (FRET) effect within the coassembly system, and the energy-transfer efficiency can reach up to 99% and 92%, which suggested

Figure 50. Modular pre- and postfunctionalization of CCMV−avidin crystals through biotin−avidin interactions. Adapted with permission from ref 233. Copyright 2014 Nature Publishing Group.

crystalline scaffolds. In summary, this work represents a new protocol for electrostatic self-assembly of native patchy proteins into ordered binary superlattices, which offers a promising scaffold for further modular and versatile functionalization with diverse biotin-tagged units. Apart from cationic polymers, AuNPs, and proteins, nonnatural dyes with specific photoactive properties represent another type of desirable inducer to dictate virus cage selfassembly into functional biohybrid crystals. Phthalocyanines (Pc) are one typical kind of unnatural organic dye with intense absorption at the near-infrared (NIR), long-lived fluorescence, and high singlet oxygen (1O2) quantum yields. However, they tend to aggregate in buffer solutions, thus losing their ability to generate singlet oxygen, which limits their potential for applications. To address this, Kostiainen et al. constructed a hierarchical protein self-assembly system where octacationic zinc Pc and tetraanionic pyrene derivatives (Figure 51a) assembly was first driven by electrostatic and π−π interactions, and the resulting cationic complex acted as a molecular glue that induced the anionic patchy apoferritin (aFt) protein cocrystallization (Figure 51b).234 The resulting ternary face-

Figure 51. Hierarchical strategy toward photoactive biohybrid crystals. (a) Chemical structures of zinc Pc and pyrene derivatives. (b) aFt cage and its further cocrystallization driven by electrostatic interactions. Adapted with permission from ref 234. Copyright 2016 American Chemical Society. 13600


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Figure 52. Schematic representation of the formation of a rmSSF linear assembly induced by PLL15. Adapted with permission from ref 235. Copyright 2014 Royal Society of Chemistry.

that a highly efficient light-harvesting system had been successfully constructed.236 Similarly, Liu and co-workers demonstrated that the corecross-linked micelles (CCMs) with cationic surfaces are also an effective inducer to guide SP1 self-assembly into 1D nanowires in a sandwich arrangement (Figure 53a).237 AFM images

as a matching acceptor chromophore and attached to the surface of CCMs. The labeling of the building blocks did not disarrange the self-assembly of the protein nanowire, and a FRET platform was constructed. The overall energy-transfer energy was calculated to be 52% from DPA to EY (path 1, Figure 53c) and 59% between adjacent donors (path 2, Figure 53c). Very recently, a simpler strategy was developed by the same group to construct highly ordered tubelike SP1 nanostructures by utilizing electrostatic interactions and small molecularethylenediamine cross-linking (Figure 54). Protein nanotubes

Figure 53. (a) Self-assembly of chromophore-labeled proteins and micelles. (b) Structural comparison of natural LH-2 complex and artificial SP1 light-harvesting system (c) Two energy-transfer paths in the FRET system. Adapted with permission from ref 237. Copyright 2016 American Chemical Society.

Figure 54. Self-assembled SP1 nanotubes. (a) SP1 nanoring. (b) Selfassembly of SP1 nanorings into protein nanotubes by multiple ethylenediamine linkers. (c) SP1-based nanotube induced by electrostatic self-assembly. (d) Nanotubes constructed by “zero-length” crosslinking. Adapted with permission from ref 238. Copyright 2016 Royal Society of Chemistry.

indicated the length of the SP1/CCMs linear structure can reach up to 200 nm. Because of the highly stable cricoid-like structure of SP1 analogous to the natural LH-2 complex of photosynthetic bacteria, it provided an optimal scaffold on which to incorporate chromophores and mimic the energytransfer process of natural LH-2 complex (Figure 53b). The Ala 84 site on SP1 was mutated to cysteine residue (Cys 84) and attached with a thiol-reactive chromophore-9-[4-(bromomethyl)-phenyl]-10-(4-methylphenyl)anthracene (DPA-Br) as a donor chromophore. Eosin Y disodium salt (EY) was employed

with highly consistent and uniform distributions were synthesized.238 Furthermore, nanoenzymes were constructed on the nanotubes by self-assembly of selenium-containing protein nanorings with glutathione peroxidase (GPx) catalytic centers. This novel strategy shows potential for the design of ideal functional nanomaterials for catalysis, biosensors, or pharmaceuticals. Moving forward, apart from the “rigid” QD inducer and “soft” micelles, Liu and co-workers further explored the possibility of utilizing other nanoparticles to induce SP1 self13601


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Electrostatic interactions have shown obvious advantages due to their long-range nature and the simplicity of control to direct protein self-assembly. This kind of driving force can be easily generated by chemical and biological methods and can be reversibly tuned by adjusting the pH value and ion strength, thus making electrostatic interactions a powerful tool to guide protein self-assembly. The reversible regulation of protein assemblies will display great potential in further preparing smart biomaterials and biodevices. Unfortunately, this strategy also has the disadvantage that the ability to fine-tune the orientation and arrangement of protein assemblies is weaker than that of other strategies due to the nonspecificity of electrostatic interactions.

assembly. Computer simulations suggested that a positively charged fifth-generation poly(amino amine) (PAMAM) dendrimer (PD5), with 128 terminal amino groups distributing around the surface and a diameter of 5.3 nm, should be another optimal inducer (Figure 55a). DLS, AFM, and TEM measure-

3.4. Host−Guest Recognition-Driven Protein Assembly

Synthetic supramolecular macrocyclic molecules, such as crown ethers, cyclodextrins, calixarenes, and cucurbit[n]urils, have been widely developed and investigated as host molecules for controllable assembly for the last two decades.242,243 Due to the selectivity, high binding affinity, reversibility, and responsiveness, a variety of supramolecular self-assembled polymeric systems have been constructed, and their collective functions in the bulk/gel states have been vastly studied.197,244,245 Besides the existing natural supramolecular interactions, the specific host−guest recognitions based on synthetic host−guest moieties open up an alternative approach to create synergistic and highly ordered protein assemblies. Among all of the host molecules, cone-shaped cyclodextrins and pumpkin-shaped cucurbit[n]urils are the most optimal platforms for biological applications due to their self-recognition behavior in aqueous media, which is fundamental for biological systems. As a classic host molecule, cyclodextrins have already been widely exploited in biochemical applications owing to their interactions with various biologically relevant guest molecules in their hydrophobic cavities.246−248 As another promising host molecule, cucurbit[8]uril (CB[8]), a cucurbit[n]uril family member with a relatively large cavity, was demonstrated to recognize a series of guest molecules through geometrical fitting and hydrophobic and ion−dipole interactions.249,250 Brunsveld and co-workers performed pioneering work with a series of protein-dimerization studies induced via host−guest interactions. The strong host−guest pair, β-cyclodextrin (βCD) and lithocholic acids (LAs), with a binding constant in the submicromolar range in aqueous media, was initially utilized as the driving force (Figure 56a).251 Two sets of cyan and yellow fluorescent protein (CFP and YFP, respectively) variants differing in the intrinsic affinities were designed and expressed as building blocks, and their dimerization was monitored by the FRET effect. When inducing the monomeric mCFP and mYFP assembly, which lacked the tendency to undergo dimerization, the resulting heterodimer exhibited a relatively low Kd of 4 × 10−6 M. However, under the same conditions, dCFP and dYFP featuring intrinsic interactions can assemble into stronger heterodimers with a higher affinity (Kd = 4 × 10−7 M). The significantly enhanced FRET effect between dCFP and dYFP suggests the positive cooperation of external host−guest interactions and innate noncovalent interactions, which may inspire the development of a means to control protein−protein interactions in order to fabricate biosensors with enhanced sensitivity. Moreover, this host−guest-induced protein selfassembly system based on β-CD and LA interactions was demonstrated to be functional in the cellular environment

Figure 55. (a) Construction of a cooperative dual-antioxidant enzymatic system. (b, c) PD5-induced protein nanotubes of SP1 via electrostatic interaction. Adapted with permission from ref 239. Copyright 2015 American Chemical Society.

ments demonstrated that PD5 can also induce the formation of one-dimensional protein nanorods (Figure 55b, c).239 Notably, the alternatively arranged protein nanorods provide a versatile platform for creating complex artificial enzymes in a cooperative manner. Very recently, focusing on the unique characteristics of SP1, the same group has utilized the thermally stable protein as scaffolds to design an efficient antioxidative glutathione peroxidase (GPx) mimic with a significant broader temperature range and high thermostability.240 However, enzymes in living organisms always function in a cooperative way: daily metabolism can produce various reactive oxygen species, among which the superoxide radical anions can only be scavenged by superoxide dismutase (SOD).241 Therefore, for long-term, effective protection against oxidative damage, it will be optimal to advance a single GPx mimic system into a synergistic GPx−SOD cooperative system. To this end, the SP1−PD5 nanorods were further utilized as scaffolds and functionalized into a dual-antioxidant system (Figure 55a). Ala 57 of SP1 located on the outer surface, which avoids being buried by PD5 particles, was mutated into selenocysteine (Sec) acting as the GPx active sites, and the substrate (GSH) was stabilized by two arginine residues (Arg 16, Arg 61). Manganese porphyrin (MnPP) as the SOD catalytic center was synthesized and linked to the amino groups of PD5 via a Michael addition reaction. Mitochondria oxidative stress assay and thiobarbituric acid (TBA) assay revealed that the cooperative antioxidant protein nanowires exert great antioxidative capacity. Moreover, as evaluated by MTT assay, 24 h incubation of enzymatic SeSP1−MnPD5 assemblies toward human lung A549 cells exhibited low cell cytotoxicity, thus demonstrating the potential of this approach for the development of functional biomaterials for catalysis, biosensors, and pharmaceuticals. 13602


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Figure 57. CB[8]−FGG interactions induced protein heterodimerization and subsequent disassembly with the addition of competing small molecules. Adapted with permission from ref 255. Copyright 2010 Wiley-VCH Verlag GmbH& Co. KGaA, Weinheim. Figure 56. (a) Supramolecular protein heterodimerization between two sets of cyan and yellow fluorescent proteins. (b) β-CD and LA interactions induced fluorescent protein self-assembly in cellular media, where the FRET effect is characterized by confocal fluorescence microscopy. Adapted with permission from refs 251 and 252. Copyright 2009 and 2007 Wiley-VCH Verlag GmbH& Co. KGaA, Weinheim, respectively.

stability of the binding pair, this protein dimerization can be reversibly disassembled by the addition of synthetic methyl viologen molecules as a competing guest molecule, which bodes great potential as a novel bioorthogonal approach for further biochemical applications. Furthermore, a tetrameric protein complex of FGG−dYFPs and FGG−dCFPs was also generated via a “dimers of dimers” pathway, which was induced by the combination of CB[8]−FGG-based host−guest interactions and predesigned intrinsic affinities.256 Beyond protein dimerization, Liu and co-workers significantly advanced the research field with the construction of extended functional protein assemblies.257 Two guest molecules, tripeptide FGGs, were genetically fused onto the Ntermini of GST homodimer in an opposite orientation. As a consequence, the resulting GST variant (FGG−GST) could readily assemble into 1D protein nanowires induced by the specific CB[8]−FGG recognition behavior (Figure 58). AFM measurements provided direct characterization of the linear morphology with an average height of 4.8 ± 0.3 nm that was consistent with the theoretical value of naturally occurring GST. A detailed investigation suggested that the spherical GST particles were divided by a spacer, which is in agreement with the CB[8] bridging mechanism. In particular, isothermal titration calorimetry (ITC) revealed a 2:1 binding ratio between FGG−GST and CB[8], and the binding constant is higher than that of sole FGG molecules and CB[8], which suggests that the interactions between the N-termini of FGG− GSTs facilitated protein self-assembly. To further functionalize this system, FGG−GST was transformed into a glutathione peroxidase (GPx) mimic with the guidance of computer simulations. The resulting GST variants can assemble into GPxfunctionalized protein nanowires, which were characterized with high stability and significant antioxidative properties for protecting mitochondria against oxidative stress.258 This work combining CB[8]-mediated host−guest interactions and enzymatic simulation not only obtained long-scale protein nanostructures but also presents an approach for further

(Figure 56b).252 This study provides a bioorthogonal way for the modulation of protein self-assembly in living organisms. Pumpkin-shaped CB[8], which can form ternary complexes with corresponding water-soluble ligands, represents an alternative way to dictate protein self-assembly. As a ligand pair forming a stable charge transfer complex inside the cavity of CB[8], methylviologen (MV) and naphthalene (Np) were chemically appended to YFP and CFP, respectively, to induce protein heteroassembly. The FRET effect validated that CB[8]/MV/Np complexes provide a powerful tool for dictating protein self-assembly and allow for specific visualization of the protein-dimerization event.253 As a versatile molecular recognition host, CB[8] can selectively associate with two tripeptide phenyalanine-glycineglycine (FGG) motifs (Kter = 1.5 × 1011 M−2),254 which can be genetically engineered into protein N-termini through molecular biology technology. Brunsveld and co-workers thus studied the induction and noncovalent reversion of CB[8]-guided FGG−mCFP and FGG−mYFP dimerization.255 A decrease in fluorescence anisotropy with the successive addition of CB[8] into a FGG−mYFP solution suggests the occurrence of a typical supramolecular recognition process. Induced protein dimerization can also be observed by size-exclusion chromatography (SEC) measurements, which suggests that the protein dimer is sufficiently stable to endure high dilution. Heterodimerization between FGG−mCFP and FGG−mYFP was monitored by fluorescence analysis that showed an increase of the peak ratio at 527 nm/475 nm from 0.46 to 2.73, which was not observed regarding two reference proteins with N-terminal methionine residues, indicating high selectivity between CB[8] and FGG motifs (Figure 57). Moreover, despite the high 13603


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the contracted state and the extended state led to a springlike motion (Figure 59c). The performance of the protein nanospring was retained after multiple switches between the contracted state and the extended state. The successful construction of this protein nanospring extends the application of functionalized protein assemblies and could be used as a new kind of nanodevice. Host−guest systems have a number of advantages for the development of protein-based nanostructures. In terms of steric structure, they simply require a small space to modify proteins without compromising the native protein configurations. Moreover, a wide variety of host−guest pairs exhibit different binding constants to satisfy the requirements for driving different protein molecules assembly. This strategy has demonstrated the superiority of manipulation when the structural information on proteins and protein−protein interactions is unknown. Additionally, the innate responsiveness and reversibility of host−guest interactions allow for the dynamic control of protein assembly/disassembly and their functionalities. Because most of the recognition events feature a strong hydrophobic effect, synthetic supramolecular host molecules may have the possibility to simultaneously recognize and bind with aromatic or aliphatic amino acids on protein surfaces with significant binding constants, which could interfere with the designed protein self-assembly processes.

Figure 58. CB[8]-induced self-assembly of FGG−GST into protein nanowires. Adapted with permission from ref 257. Copyright 2013 Wiley-VCH Verlag GmbH& Co. KGaA, Weinheim.

functionalization of protein nanostructures with advanced properties. Developing stimuli-responsive biomaterials on assembled proteins has attracted increasing attention. Nanospring, which can perform springlike motions with controllable functions, can act as smart biomimetic muscle tissue materials. By using the same strategy, Liu and co-workers developed a self-assembled protein nanospring through host−guest interactions between CB[8] and tripeptide FGG tags of fusion protein FGGrecoverin-GST (Figure 59a, b).259 This nanospring displayed a structural change in response to Ca2+, and the change between

3.5. Polymer−Protein Conjugates Self-Assembly

The approaches to prepare polymer−protein conjugated nanomaterials can be broadly divided into two main classes: (i) Polymers preform into ordered nanostructures onto which proteins are subsequently decorated. This approach usually needs to incorporate specific interacting moieties onto polymer surfaces for specific recognition of proteins, which will be discussed in section 3.6.2 of this Review. (ii) Protein−polymer conjugates are initially synthesized and subsequently undergo directed self-assembly into ordered nanostructures in a bottomup manner. Here, we mainly focus on recent advances on protein−polymer conjugates self-assembly systems of the second class, highlighting ordered architectures and discussing their stimuli-responsiveness for potential applications in therapeutics,260 biosensors,261 drug delivery,262 and biochips.263 In most protein−polymer conjugate-based self-assembly systems, the modules behave as “giant amphiphiles” and undergo self-assembly via hydrophobic interactions. Significantly, the study should begin with the strategies for “grafting” the hydrophobic polymer part to the hydrophilic protein part to allow the construction of biohybrid giant amphiphiles. Going deeper, the strategies can be further sorted into two classes according to the nature of the binding forces: (i) linking via noncovalent recognitions or (ii) linking via covalent chemical reactions. 3.5.1. Non-Covalently Linked Polymer−Protein Conjugates Self-Assembly. As for noncovalent linking strategies, naturally selective receptor−ligand interactions such as streptavidin−biotin interactions and enzyme−cofactor interactions are the most commonly used supramolecular tools to prepare protein−polymer conjugates that self-assemble in a completely new manner. Nolte and co-workers first prepared a hybrid amphiphilic system via the recognition of biotinylated polystyrene (PS) with streptavidin.264 The biotinylated PS chains were spread at the air/water interface to form a monolayered rigid film, and they were subsequently incubated with streptavidin. The specific binding of two biotinylated PS

Figure 59. (a) Recombinant plasmid of FGG-recoverin-GST; (b) FGG-recoverin-GST dimer; (c) contracted and extended states of a host−guest-induced protein nanospring controlled by Ca2+. Adapted with permission from ref 259. Copyright 2016 Royal Society of Chemistry. 13604


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chains to one side of the streptavidin binding pocket was validated by AFM characterizations and confocal fluorescence microscopy. This unique binding mode with two available binding sites allows for further functionalization via other biotinylated moieties. The PS−streptavidin conjugates can also be enzymatically functionalized with HRP, whose catalytic activity was notably retained. Hoffman, Stayton, and co-workers also performed a series of protein−polymer conjugates studies based on streptavidin modules focusing on the ligation of “smart” polymers, which endow reversible control of the system via external stimuli such as temperature and pH value. For example, reversible control of protein−polymer particle sizes was demonstrated via the bioconjugation of biotinlyated temperature-responsive polymer poly(N-isopropylacrylamide) (PNIPAM) with streptavidin.265 Below the lower critical solution temperature (LCST), PNIPAM is hydrophilic and the conjugates remain soluble. When the temperature is above the LCST, PNIPAM turns hydrophobic and aggregates into nanoparticles with defined sizes with streptavidin acting as a stabilizer to prevent further aggregation (Figure 60). The size of the nanoparticles can be

Figure 61. (a) General concept to construct protein−polymer conjugates via the cofactor-reconstitution method. Adapted with permission from ref 267. Copyright 2009 American Chemical Society. (b) SEM images of the aggregates of ferriprotoporphyrin IX-modified PS chain. (c) TEM images and (d) cryo-TEM images of the PS−HRP biohybrid conjugates self-assembly nanostructure. Bars represent 200 nm. Adapted with permission from ref 268. Copyright 2002 WileyVCH Verlag GmbH& Co. KGaA, Weinheim. Figure 60. Schematic representation of the temperature-dependent formation of PNIPAM−streptavidin smart nanoparticles. Adapted with permission from ref 265. Copyright 2004 American Chemical Society.

cofactor (ferriprotoporphyrin IX)-modified PS polymer chains.268 In the absence of the apoenzyme, the synthesized polymer can aggregate into spherical aggregates with diameters of 100−1000 nm and perforated wall structures (Figure 61b). However, the addition of a tetrahydrofuran (THF) solution of the polymer to an aqueous solution containing excess apoenzymes can result in vesicular aggregates with diameters of 80−400 nm (Figure 61c, d). Most of these aggregates enclosed spherical objects that were often isolated from the center of the aggregates. A plausible mechanism for this phenomenon is that the heme-functionalized polymers first form aggregates prior to the reconstitution reaction of the apoHRP; the latter formation of the biohybrids self-assembles into vesicles and thus encapsulates the initial aggregates. Enzymatic assay revealed that the bioconjugates exhibited no activity at 4 °C. However, when the reconstitution was carried out at 22 °C, the hybrid material surprisingly regained much of its original activity. This work demonstrated for the first time the use of a cofactor-reconstitution method to yield giant amphiphiles that can self-assemble into aggregates with catalytic activity in aqueous media. Similarly, with this method, another heme-binding protein, myoglobin (Mb), was also utilized to conjugate with cofactorlabeled PS chains to form giant amphiphiles, which can also self-assemble into well-defined spherical aggregates.269 The stability of the dioxygen myoglobin complex within the hybrid is reduced when compared with native Mb, which may attribute to disturbed binding of the heme in the apoprotein.

controlled across a mesoscale range of ∼250−900 nm, which can be manipulated through the polymer molecular weight, the concentration, and the heating rate. Moreover, in comparison with PNIPAM aggregates, the conjugated particles are remarkably stable, and their reversible assembly/disassembly can be feasibly controlled. Similarly, Stayton and co-workers prepared a biotinterminated reversible addition−fragmentation chain transfer (RAFT)-based copolymer composed of PNIPAM-b-PAA (PAA = poly(acrylic acid)) blocks.266 The conjugates of this copolymer and streptavidin exhibited distinct self-assembly behavior: the thermally induced aggregation and phase separation of PNIPAM−streptavidin conjugates was prevented through shielding of the hydrophilic PAA block. In addition, the cloud point and aggregation properties of the copolymer− streptavidin conjugates exhibited pH-dependent behavior that differed significantly from that of free copolymers. Apart from streptavidin−biotin interactions, apoenzyme− cofactor interactions have also been widely applied to prepare noncovalent protein−polymer conjugates. Nolte and coworkers proposed a general concept (Figure 61a) where the natural cofactor of the enzyme was removed prior to the reconstitution with the synthesized cofactor end-capped polymer to construct giant amphiphiles.267 They began with the demonstration of the conjugation of HRP enzymes with 13605


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architectures with a complexity approaching those found in Nature. 3.5.2. Covalently Linked Polymer−Protein Conjugates Self-Assembly. Another typical method to link polymer components to protein components is via covalent bonds. However, considering that proteins tend to be complex and fragile reagents, the chosen chemical reaction should be specific, highly efficient, and capable to be performed under mild conditions in aqueous media. Over the years, the toolkit of bioconjugation reactions has continued to expand. Methods for modifying the amino acids on protein surfaces typically target the side-chains of lysine, glutamate, aspartate, and cysteine (Scheme 3).272,273 Among all of these amino acids, cysteine is the most typical targeting amino acid attributing to its relatively low abundance and high reaction activity toward maleimides, disulfides, and iodoacetamides under near physiological conditions.274 In particular, the relatively low surface abundance of cysteine enables site-specific surface modification that is facilitated by genetic mutation. The cysteine alkylation with maleimides has stood out as the most widely adopted and broadly successful approach. For example, Nolte and co-workers chose the lipase B from Candida antarctica (CALB) as the hydrophilic head and a maleimide-functionalized PS of 40 repeat units as the hydrophobic tail.275 To conjugate these two components, a disulfide bridge positioned on the outer surface of CALB was reduced to produce two free thiol groups, one of which was subsequently connected with the maleimide functional group. The resulting giant amphiphiles can self-assemble into micrometer-long fibers built up from bundles of micellar rods. The smallest rod exhibited a diameter of 20−30 nm, which closely corresponded to the predicted diameters of the micellar architectures (Figure 63). On the basis of the thiol−maleimide coupling approach, Olsen and co-workers performed a series of studies utilizing covalently linked mCherry−PNIPAM as model biohybrid copolymers.276 The particular emphasis of these investigations was focused on the underlying mechanism of self-assembly in concentrated solutions and the resulting solid-state nanostructures. They began with the utilization of a red fluorescent mCherry protein variant mCherryS131C with a unique cysteine residue for precise ligation with PNIPAM.276 The self-assembly process was induced by solvent evaporation, and due to the thermoresponsive nature of PNIPAM, water at different temperatures can act as a selective as well as a nonselective solvent. Above the LCST, PNIPAM became hydrophobic and thus only proteins were selectively dissolved by water. Below the LCST, PNIPAM was fully hydrated and thus water acted as a nonselective solvent for the solution. The influence of this transition on the self-assembly behavior was obvious. At 40 °C, the bioconjugates self-assembled into a heterogeneous nanodomain structure, whereas a hexagonal perforated lamellar phase was observed at room temperature. Subsequent solvent annealing gave rise to well-ordered lamellar structures, which may be closer to thermodynamic equilibrium (Figure 64). Further studies revealed that the self-assembled morphologies of the mCherry−PNIPAM diblock can be manipulated into cylinders, perforated lamellae, lamellae, or hexagonal and disordered micellar phases depending on the polymer fraction and the solvent selectivity.277 Notably, a large majority of the protein tertiary structure and function can be preserved in these

Following the same strategy, Nolte and co-workers further explored the change of the system engendered via variation of the polymer component. To this end, cofactor-functionalized synthetic diblock copolymer polystyrene-b-polyethylene glycol (PSm-b-PEG113) was conjugated with Mb or HRP, respectively, to form biohybrid ABC triblock copolymers.270 The obtained amphiphilic conjugates can self-assemble into a large variety of nanostructures (Figure 62). As previously mentioned, amphi-

Figure 62. Morphologies of triblock copolymers of Mb-b-PS-b-PEG and HRP-b-PS-b-PEG self-assembly in water: (A, B) toroids; (C) schematic figure of a toroid; (D) octopi; (E) figure eights; (F−H) micellar aggregates. (I) SEM images of aggregates of MbZn-b-PS48-bPEG113, which form spherical aggregates consisting of lamellae. Bars represent 100 nm for panels A−H and 500 nm for panel I. Adapted with permission from ref 270. Copyright 2007 American Chemical Society.

philic diblock copolymers containing hydrophobic PS chains and a HRP or Mb hydrophilic head can predominantly selfassemble into spherical aggregates.268,269 Addition of a third hydrophilic block to the diblock copolymer significantly changed the self-assembly behavior of the system. With the same cofactor-reconstitution concept, recently Wan and Liu achieved thermal control of the biohybrid self-assembly system by coupling the typical thermoresponsive PNIPAM polymer with Mb.271 The functionalization of the porphyrin cofactor to the polymer was achieved with the combination of atom-transfer radical polymerization (ATRP) and click reactions. The obtained biohybrid diblock copolymer selfassembled in a typical thermoresponsive manner: Below the phase-transition temperature of the PNIPAM segment, the hybrid remained hydrophilic; above the phase-transition temperature, the hybrid self-assembled into PNIPAM-core micelles that were stabilized by the hydrophilic Mb coronas. The self-assembly of giant amphiphiles is still a relatively new field in supramolecular chemistry. This approach, however, opens new avenues for the construction of functional biomimetic assemblies. Using proteins, enzymes, and other protein aggregates as hydrophilic groups provides unique possibilities for the construction of functional protein nano13606


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Scheme 3. Bioconjugation Methods for the Modification of Protein Surfaces via Covalent Linkage (Adapted with Permission from Ref 272; Copyright 2015 American Chemical Society)

Figure 63. Synthesis of covalently linked CALB−PS giant amphiphiles that self-assemble into micellar rods in water. Adapted with permission from ref 275. Copyright 2002 American Chemical Society.

Olsen and co-workers also conjugated the PNIPAM chains to a functional Mb moiety.279 The resulting Mb−PNIPAM copolymers self-aggregate into weakly ordered lamellar domains at a concentration of 50 wt % and above. In comparison with previous mCherry−PNIPAM and GFP− PNIPAM conjugates, Mb−PNIPAM conjugates are poorly ordered, which confirmed that changes in protein structure can result in large changes in their self-assembly behavior. However, the structure and activity are well-conserved in this conjugate, which can be coated onto silicon support to fabricate a highly active biocatalytic film. On a protein basis, this bioconjugate film performs 5−10 times better than catalysts made from other commonly used enzyme-immobilization strategies. Recently, in an elegant study, Mann and co-workers presented a general approach to prepare protein−polymer hybrid bioconjugates that can self-assemble into large microcompartments termed as proteinosomes at the oil/water interface.280 First, the giant amphiphiles were synthesized via covalently label amine groups on bovine serum albumin (BSA)

self-assembled materials, which may open up new routes toward functional biomaterials. The effects of variation of the protein “head” on the overall self-assembly behavior were also investigated by comparing mCherry−PNIPAM and green fluorescent protein (EGFP)− PNIPAM bioconjugates.278 mCherry and GFP are both structurally similar β-barrel shaped proteins, albeit with significantly different chemical compositions and surface potentials. Experiments suggested that they are both able to form nanostructured solid-state materials (Figure 65). The similar self-assembly behavior of the two conjugates suggested that coarse-grained properties such as the protein shape, solubility, charge, and virial coefficient are largely responsible for the self-assembly behavior of the protein−polymer conjugates. However, variation of the protein “head” does engender some differences: desolvation occurs with mCherry− PNIPAM, which exhibits a wide two-phase region, whereas GFP−PNIPAM forms homogeneous disordered micellar solutions. 13607


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PAAm nanoconjugates that yield similar proteinosomes. Interestingly, these large, stable proteinosomes provide great scaffolds for the construction of artificial cells. As a paradigm, an in vitro expression system of GFP has been constructed by adding synthesized pEXP5-NT/eGFP plasmid DNA vectors to a cell-free gene-expression solution (Figure 66e). In vitro expression of eGFP, especially within the proteinosomes, was validated by fluorescence microscopy (Figure 66f, g). Significantly, this study provides a general approach to prepare protocells with a range of biomimetic properties including guest-molecule encapsulation, selective permeability, protein synthesis via gene expression, and membrane-gated internalized enzyme catalysis. In recent years, with the development of engineering unnatural amino acid residues onto protein surfaces, apart from the modification of traditional natural amino acids, selective modification methods targeting unnatural amino acids have become recognized as new routes for the synthesis of protein−polymer conjugates. Matyjaszewski and co-workers engineered GFP surfaces with reactive azide moieties via sitedirected mutagenesis.281 The resulting GFP−diN3 variants were directly copolymerized with monodispersed dialkyne-terminated poly(ethylene oxide) (PEO−dialkyne) via click reactions under mild aqueous conditions (Figure 67). Notably, the degree of polymerization (DP) of PEO was defined to be 20 and the polymer component here actually acted as flexible linkers. Confocal microscopy and AFM and DLS characterization revealed that the covalently linked copolymers selfassembled into uniform, highly persistent fibers with lengths on the micrometer scale. This self-assembly behavior was ascribed to the tendency of GFP molecules to dimerize through localized hydrophobic patches and flexible PEO chains. With a similar click reaction toward unnatural amino acids, Jewett, O’Reilly, and co-workers constructed a thermoresponsive protein−polymer self-aggregate system.282 Azide-functional amino acids p-azidophenylalanine were functionalized onto GFP surfaces at different positions and subsequently linked with alkyne-capped poly[(oligoethylene glycol)methyl ether methacrylate] (PEGMA) polymers. The resulting bioconjugates retained their innate fluorescence. Turbidity measurements revealed that all three bioconjugates form aggregates with increasing temperature, which suggested that the conjugating sites do not influence the shape of the bioconjugate (Figure 68). However, the transition temperature was found to be dependent on the molecular weight of the polymer and polymer-conjugation locations. Significant attention has also been devoted to the conjugation of thermally responsive elastin-like polypeptide (ELP) to protein surfaces. Because of the polypeptide nature of ELP, they can be incorporated via genetic approaches; thus, the studies do not strictly fall in the category of the bioconjugates of natural “protein” moieties and synthetic “polymer” moieties. However, ELPs can switch from an extended water-soluble state to a collapsed hydrophobic state in response to an increment in temperature. Thus, the self-assembly of conjugates of ELP−protein resembles that of traditional protein−polymer bioconjugates. In addition, ELP−protein conjugates have innate biocompatibility, which may endow them with wider potential for applications, particularly in vivo applications. For example, Cornelissen and co-workers constructed a viral protein self-assembly system with two self-assembly mechanisms that were governed by the pH and temperature, respectively.283 As an excellent example of natural reversible

Figure 64. Two possible pathways toward self-assembly of mCherry− PNIPAM bioconjugates. Room-temperature water provides a nonselective solvent, whereas at 40 °C water acts as a protein-selective solvent. Adapted with permission from ref 276. Copyright 2011 American Chemical Society.

Figure 65. mCherry−PNIPAM and GFP−PNIPAM conjugates exhibit similar self-assembly behavior such as forming the lamellar phase. Adapted with permission from ref 278. Copyright 2014 American Chemical Society.

surfaces with mercaptothiazoline-activated PNIPAM polymers (Figure 66a). The resulting BSA−NH2−PNIPAM conjugates can self-assemble into proteinosomes with diameters in the range of 25−50 μm (Figure 66b). The obtained proteinosomes exhibited great stability to remain in an aggregated state at room temperature over several weeks, and their robust ultrathin membrane structure even remained intact upon vacuum evaporation (Figure 66c, d). In addition, this methodology is versatile and can be employed with various building blocks such as myoglobin−NH2/PNIPAAm or hemoglobin−NH2/PNI13608


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Figure 66. (a) Schematic representation of the synthesis route of the BSA−NH2/PNIPAM conjugates that self-assemble into proteinosomes at the oil/water interface. (b) Optical microscopy images of proteinosomes dispersed in oil. (c) AFM and (d) TEM images of a dried proteinosome. (e) Schematic representation of encapsulation and in vitro gene expression of GFP within proteinosomes. (f) Optical microscopy image of proteinosomes containing GFP gene-expression system after 2 h of incubation at 37 °C and (g) corresponding fluorescence micrograph images validating the expression of GFP (scale bar, 100 mm). Adapted with permission from ref 280. Copyright 2013 Nature Publishing Group.

Figure 67. Schematic representation of the click step-growth polymerization approach for preparing GFP−PEO copolymers. Adapted with permission from ref 281. Copyright 2014 Wiley-VCH Verlag GmbH& Co. KGaA, Weinheim.

Figure 68. Schematic representation of the synthesis of GFP−PEGMA bioconjugates with different modification sites. As the temperature increased, all conjugates self-aggregated. Adapted with permission from ref 282. Copyright 2015 American Chemical Society.

nanomachines, CCMV can perform pH-dependent assembly/ disassembly: at high pH (7.5), empty CCMV dissociates into capsid protein (CP) dimers, and they reassemble at low pH (5.0).284 Through integration of the pH-responsive CCMV moiety with the temperature-responsive ELP moiety via genetic fusion, the resulting fused ELP−CPs can self-assemble into two different, well-defined nanocapsules via dual mechanisms: pH induced self-assembly into relatively large virus-like particles (28 nm) and ELP-induced assembly into 18 nm nanocapsules (Figure 69). In a separate study, Chilkoti and co-workers genetically fused a set of ELP block copolymers differing in hydrophilic and hydrophobic block lengths to two protein domains, thioredoxin (Trx) and afibronectin type III domain (Fn3).285 Analogous to traditional giant amphiphiles, the resulting conjugates can selfassemble into spherical micelle particles in response to

Figure 69. Schematic representation of ELP−CCMV conjugate selfassembly into two different virus-like particles with different diameters in response to dual stimuli. Adapted with permission from ref 283. Copyright 2012 American Chemical Society.

increments in the solution temperature (Figure 70). The variation of the protein “head” was characterized to have an 13609


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Figure 70. Schematic representation of the expected self-assembly behavior of protein−ELP fusion conjugates. Adapted with permission from ref 285. Copyright 2012 American Chemical Society.

AuNPs, which are dictated by the heme−heme pocket interactions, can be formed (Figure 71).286

effect on the micelle-forming temperature via influencing the inverse transition temperature of the hydrophilic block. As mentioned before, due to the innate biocompatibility of ELP, the ELP fused protein bears great potential to express and self-assemble in vivo. Schiller and co-workers managed to genetically program and perform de novo synthesis of amphiphilic proteins to form intercellular compartments,174 which is described in section 2.5 of this Review. Synthetic polymers exhibit diverse properties such as hydrophilicity/hydrophobicity and stimuli-responsiveness that can be employed to direct protein assembly through polymer− protein conjugate formation. This combinatorial method can provide increased complexity and bioorthogonal control for the construction of more advanced biomaterials. Due to the bioconjugation relying upon chemical modification of the reactive groups on protein surfaces, the drawback of reduced biological activity is frequently encountered in the preparation of polymer−protein conjugates. Current efforts are devoted toward developing mild and efficient reactions to achieve sitespecific protein modifications for bioactive protein assemblies.

Figure 71. Preparation of AuNPs and hemoprotein hybrids via receptor−ligand interactions. Adapted with permission from ref 286. Copyright 2010 Royal Society of Chemistry.

3.6. Chemical Template-Induced Protein Assembly

Utilizing predetermined ordered nanostructures as templates is an efficient way to assemble various proteins in a predictable manner, leading to more complex protein aggregates and allowing for the facile construction of functional biomaterials. The general concept of templating is guiding the self-assembly of proteins into aggregate forms via the specific recognitions with pre-existing, well-defined templates. The essential interactions between chemical templates and protein building blocks are always accomplished via supramolecular interactions such as receptor−ligand interaction, electrostatic interactions, and metal coordination. However, in this category the ordered nanostructures of the templates play central roles without which the protein-assembly system cannot be constructed, which distinguishes these from the earlier-discussed proteinassembly systems that are dictated directly by supramolecular interactions. In this section, the discussion of recent developments in this area is organized around the various types of utilized chemical templates. 3.6.1. Nanoparticles as Templates. Nanoparticles have innate electronic, magnetic, and photonic properties. Typically, gold nanoparticles (AuNPs) are the most widely adopted nanoparticles serving as templates to guide protein selfassembly. Hayashi and co-workers extended the application of receptor−ligand interactions via functionalizing nanoparticles with chemically modified ligands to form hybrid protein− nanoparticle complexes. Upon the modification of gold nanoparticles with covalently immobilized heme moieties, dendrimer-like supramolecular structures or densely gathered

Metal-chelating motifs can also be modified onto AuNP surfaces to guide controlled protein self-assembly. In an alternative study,287 Wang and co-workers decorated AuNPs with Ni−NTA chelates; while monofunctionalizing a His-tag on the external surface of Dps (DNA binding protein from starved cells) nanocages, with the aid of metal coordination, protein nanocages can hierarchically self-assemble into discrete nanoarchitectures (Figure 72) that are governed by the stoichiometric ratio of the building blocks and the size of the AuNPs.

Figure 72. Schematic illustration of the AuNP-templated assembly of discrete nanoarchitectures of Dps and corresponding TEM images. Adapted with permission from ref 287. Copyright 2015 American Chemical Society. 13610


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and cryo-ET.291 AuNPs with an average diameter of 11.3 nm have been used as model templates for the self-assembly of BMV capsid proteins at different concentrations. The VLP selfassembly mechanism involves an initial rapid formation of large nanoparticle−protein clusters, which subsequently bud off and separate into individual VLPs (Figure 74). This mechanism is

Apart from modification of AuNP surfaces with discrete ligands for protein recognition, protein coatings around AuNP cores represent another typical approach to prepare templated protein self-assemblies. Dragnea and co-workers demonstrated an efficient route to synthesize ordered virus-like particles (VLPs) with brome mosaic virus (BMV) protein coatings and PEG-functionalized AuNP cores.288 The PEG chains on the AuNPs were able to mimic the electrostatic behavior of the nucleic acid component of the native virus, which facilitates the efficient coating of capsid proteins. The resulting VLPs are highly homogeneous and symmetric with stoichiometry and packing properties that exhibit great similarity to native BMV viruses. Moreover, a possible mechanism was proposed for VLP self-assembly: first, electrostatic interactions lead to the formation of disordered complexes, which is followed by the subsequent formation of a crystallization phase that is guided by protein−protein interactions to form regular VLPs (Figure 73a).

Figure 74. Schematic representation of the kinetic self-assembly pathway of VLPs. Adapted with permission from ref 291. Copyright 2013 American Chemical Society.

highlighted with the remarkable selectivity of the coating proteins that can program the self-assembly events even in the crowded environment encountered within the protein−nanoparticle aggregates, which exhibited astonishing similarity with the assembly robustness of natural virus capsid proteins in crowded cytoplasm. 3.6.2. Polymers as Templates. Polymers, which can selforganize into various organized structures, represent another ideal template for guiding protein self-assembly. Wooley and co-workers prepared biotin-functionalized shell cross-linked nanoparticles via the co-micellization of chain terminal biotinylated poly(acrylic acid)-b-poly(methyl acrylate) (PAAb-PMA) and nonbiotinylated PAA-b-PMA (Figure 75).292 The obtained nanoparticles can template avidins packing at the surface via specific receptor−ligand interactions. Moreover, the multivalency of surface-available biotin can be tuned by varying the stoichiometric ratios of the biotinylated versus the nonbiotinylated blocks. Periodically branched dendrimer molecules have also shown great potential to template protein self-assembly to form hybrid protein nanoparticles. In a recent study, Escosura, Torres, and co-workers designed negatively charged phthalocyanine (Pc) dendrimers, around which the self-assembly of capsid proteins of CCMV can form into 18-nm virus-like nanoparticles.293 The density of surface charges depends on the dendrimer generation, whereas Pc aggregation can be tuned via the Pc metal center and its availability for axial substitution. The systematic study revealed that both parameters influence the outcome and efficiency of the templated self-assembly process (Figure 76). 3.6.3. Supramolecular Aggregates as Templates. Synthesized supramolecular frameworks provide extended scaffolds with attractive characteristics such as dynamic behavior, tunability, and reversibility, which allow for their adaptation as flexible templates for protein self-assembly. As a typical example, Brunsveld and co-workers reported an autofluorescent discotic monomer featuring a large planar aromatic core and peripheral ethylene glycol chains decorated with biotin motifs, which can further immobilize streptavidin or antibiotin antibodies.294 In water, the discotic monomer can self-assemble into highly fluorescent columnar structures via π−π stacking. In addition, protein self-assembly along the

Figure 73. (a) Proposed mechanism of VLP self-assembly. Adapted with permission from ref 288. Copyright 2006 American Chemical Society. (b) TEM images of obtained VLP 2D lattice, Fourier transforms (inserts), and corresponding Fourier projection maps. Adapted with permission from ref 289. Copyright 2007 National Academy of Sciences.

In a subsequent systematic study, it was revealed that the gold core diameter provides control over the capsid structure: the required coating subunits increased with the core diameter.289 VLPs with varying diameters were found to resemble three classes of viral particles found in cells. Notably, due to their high regularity, under the same crystallization conditions as the natural wild-type virus, VLPs can selfassociate into large 2D crystals within which the Au core, the PEG shell, and the protein capsid can be identified (Figure 73b). Furthermore, the influence of surface charge of the AuNP core on the self-assembly system were decoupled with the coresize variables and investigated in detail.290 It was revealed that a critical charge density is required to accommodate the selfassembly processes. Moreover, experiments suggested that the yield of encapsulation rather than the size and morphology of the VLPs is dependent on the variation of surface charges. In particular, the highest charge density gave the best yield of complete VLPs. Malyutin and Dragnea further investigated the kinetic selfassembly pathway of the VLPs via time-course light scattering 13611


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Figure 75. Schematic representation of the preparation of biotinylated polymer nanoparticles. Adapted with permission from ref 292. Copyright 2004 American Chemical Society.

Dynamic intermixing of two sets of protein-conjugated discotic polymers displayed a time-dependent increase in FRET, which indicates the occurrence of the dynamic exchange and rearrangement between the supramolecular polymers leading to mutual proximity between fluorescent proteins. This postassembly modulating protocol provides an entry for the construction of fine-tuned heterovalent supramolecular structures. 3.6.4. Templated Protein Assembly via Layer-byLayer Strategy. Layer-by-layer strategy represents another alternative way to fabricate protein assemblies. We know that the wetting of porous templates with polymer solutions is a simple and versatile approach to organize the building block into defined tubular architectures.296 However, as a prerequisite, the pore walls of the templates must have a high surface energy for the formation of mesoscopic films of the ambient solutions rather than completely filling the nanopores. Inorganic oxides meet this requirement, and porous aluminum oxide has become the most utilized porous template for the preparation of tubular nanomaterials. The tube walls can be made of a multitude of materials, and previous works have mainly focused on the construction of templated polymer nanotubes.296 Recently, this template strategy has been extended to the preparation of protein nanotubes combined with layer-by-layer deposition strategy. The general fabrication procedure is schematically summarized in Figure 78.297 In one example, Li and co-workers synthesized protein nanotubes via alternatively absorbing human serum albumin (HSA) proteins with opposite charges (by tuning the pH) or sequentially absorbing HSA and L-R-dimyristoylphosphatidic acid (DMPA), respectively, using a porous anodic alumina substrate as the template.298 SEM images revealed that both self-assembly conditions can give rise to highly ordered protein nanotubes with a monodispersed size distribution and a uniform orientation (Figure 79). Moreover, the sizes, shapes, and structural properties of the nanotubes can be controlled by the templates. Biologically active proteins have also been used to construct functional protein assemblies. Similarly, with the combination of the template method and the layer-by-layer method, Li and co-workers further synthesized protein nanotubes composed of cytochrome C with glutaraldehyde or polystyrene sulfonate (PSS).299 The assembled nanotubes were characterized to possess a uniform size and flexible shape. Notably, it was found

Figure 76. Dendrimer-templated self-assembly of CCMV CP into 18nm biohybrid VLP. ZnPc1 and RuPc2 are efficient templates, whereas only a small proportion of particles is obtained in the presence of ZnPc2 and RuPc1. Adapted with permission from ref 293. Copyright 2015 American Chemical Society.

biotinylated supramolecular polymeric scaffolds was confirmed using FRET between discotic donor fluorophore and Cy3 labeled SA. Further interprotein interactions along the supramolecular templates were investigated via utilizing SA variants labeled with Alexa Fluor 633 (SA-633) and Texas Red (SA-TR). FRET analysis indicated that the donor fluorescence of SA-TR was lost and the signal of SA-AF633 remarkably increased, which suggests that protein molecules are densely packed and in close proximity with each other (Figure 77). As an extended study, Brunsveld and co-workers utilized a similar discotic molecule, featuring trivalent functionalization with an O6-benzylguanine moiety for covalent conjugation with SNAP-tag-fused fluorescent proteins (YFP-SNAP and CFPSNAP), as a platform to investigate the dynamic and selfregulatory properties of the supramolecular assembly.295 13612


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Figure 77. Self-assembly of fluorophore-labeled streptavidin by supramolecular templates for FRET. Adapted with permission from ref 294. Copyright 2011 Royal Society of Chemistry.

Figure 78. Schematic diagram of the protein nanotube fabrication process by a layer-by-layer deposition strategy. Adapted with permission from ref 297. Copyright 2008 Springer Science+Business Media B.V. Figure 79. (a, b) SEM images of DMPA/HSA nanotubes and (c, d) HSA nanotubes obtained by template-based protein self-assembly. Adapted with permission from ref 298. Copyright 2005 American Chemical Society.

that the assembled protein nanotubes retained the electronic and bioactive properties of the building block. Martin and co-workers also managed to synthesize protein nanotubes with enzymatic activity: glucose oxidase protein was chosen as the model protein and was alternately absorbed onto the templates with glutaraldehyde as the cross-linking agent.300 The number of protein nanotube layers can be controlled by varying the alternating cycles. It was revealed that the glucose oxidase within the nanotubes retained their enzymatic activity, which may be even further enhanced as the wall thickness increases. Likewise, Tsuchida and co-workers demonstrated another study to construct functional protein nanotubes, in this case highlighting the artificial incorporation of desired enzymatic activity into the system.301 In particular, the nonspecific binding property of HSA has been utilized to complex with a synthetic

heme−FeP to form an artificial hemoprotein that can reversibly coordinate with O2. Alternatively, the adhesion of HSA−FeP complexes at low/high pH values gave rise to highly ordered HSA−FeP nanotubes (Figure 80). The O2 binding affinity of the nanotubes was 2-fold lower than that of the monomeric HSA-FeP, which was kinetically unstable due to the low association rate constants. However, this work still demonstrated a new synthetic strategy via hybridizing with desired functional modules, which will lead to the development of novel functional protein nanostructures with not only enhanced biological properties but also electronic, photonic, and magnetic performance. 13613


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reversibly form disulfide bonds under oxidation−reduction control. Moreover, as mentioned before, given the fact that cysteine is a rare amino acid, specific modification of which via thiol−maleimide bioconjugation chemistry has been widely applied.303 For instance, Mougous and co-workers reported the construction of protein nanotubes through disulfide-bondenhanced steric and chemical complementary of the homosexameric ring-shaped Hcp1 variant (Figure 81).304 The length

Figure 80. SEM images of HSA−FeP nanotubes: (a) micrometer-scale length, (b) isolated and flexible structure, (c) perpendicular orientation on the silicon wafer and the uniform outer/inner diameters, (d) section of the nanotubes and very smooth surface. Adapted with permission from ref 301. Copyright 2007 Royal Society of Chemistry.

Figure 81. Structure-based design of Hcp1 nanotubes through an intermolecular disulfide bridge between C90 and C157. (a) Overview of the Hcp1 hexameric ring structure. Hcp1 variant prepared by mutation of Arg157 and Gly90 on the top and bottom faces into cysteine for covalent protein self-assembly. (b) Hcp1 honeycomb crystal lattice (PDB: 1Y12) viewed perpendicular to the crystallographic z-axis. (c) Model of an Hcp1 nanotube. Adapted with permission from ref 304. Copyright 2008 National Academy of Sciences.

Apart from the traditional anodic aluminum oxide templates, Komatsu and co-workers utilized track-etch polycarbonate (PC) membranes as the nanoporous templates.302 The PC membrane allows for rapid dissolution with CH2Cl2, which provides a general procedure to produce a variety of protein nanotubes without the need for interlayer cross-linking. On the basis of this strategy, well-defined poly-L-arginine (PLA)−HSA, PLA−ferritin, and PLA−Mb nanotubes were synthesized. It was revealed that the outer diameters of the nanotubes can be controlled by adjusting the pore sizes of the templates, whereas the wall thickness of the nanotubes depends on the globular size of the protein (ferritin > HSA > Mb). In comparison with other templated protein assembly strategies, the layer-by-layer strategy demonstrated a convenient way for preparing largescale protein biomaterials. Chemically synthesized molecules, including inorganic and organic constructs, are robust frameworks for the creation of multidimensional high-density protein patterns. In this strategy, template-directed self-assembly of proteins through covalent and noncovalent immobilization to form hybrid nanostructures can achieve a high level of control and freedom and simultaneously ensure the structural integrity and proper orientation of protein components for functional applications. Owing to the fascinating properties and tunable structures of template molecules, the sizes, shapes, and distributions of protein assemblies can be regulated by altering the synthetic conditions. However, in the case of some complex selfassembling systems, chemical templates often involve timeconsuming synthetic procedures.

of the obtained nanotubes can be controlled through chain termination via adding specific single-cysteine-functionalized Hcp1 variants. Moreover, the polarity of the nanotube can be distinguished by the incorporation of distinct capping units. Furthermore, preliminary studies focusing on the modification of the interior of the nanotubes with thiol-reactive dendrimers provide further development opportunities for nanotubes as a nanoencapsulation system. The concept of utilizing disulfide bonds to direct the selfassembly of cyclic protein oligomers into protein nanotubes has been further investigated. Heddle and co-workers produced a self-assembled nanotube utilizing an 11-mer cyclic, thermostable TRAP (trp RNA-binding attenuation protein) variant (Figure 82a).305 In this case, two mutations of the V69C and E50C that are located on opposite faces orthogonal to the central ring axis were conducted to connect two rings via the formation of intermolecular disulfide bonds. As characterized by TEM, the TRAP variants readily self-assemble into long protein nanowires and form bundles in the presence of dithiothreitol (DTT) (Figure 82b, c). DLS analysis performed during the nanotube formation indicated that the hydrophobic interactions may also contribute to the stabilization of the protein nanotubes. Moreover, single-particle reconstruction analysis of the TEM images revealed that the TRAP variants aligned in a “head-to-head” pattern within the protein nanotubes. Another elegant work was demonstrated by Tsumoto and coworkers, in which the disulfide bonds were utilized to control the covalent protein self-polymerizing process.306 To provide

3.7. Covalent Protein Self-Assembly

Most works reported in this Review involve supramolecular interaction-based protein self-assembly. However, beyond the scope of supramolecular chemistry, based on direct chemical cross-linking strategies, some typical protein nanostructures have also been created. Notably, most protein covalent crosslinking systems rely on the cysteine residue, which can 13614


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Further investigation into the self-assembling mechanism was studied through utilizing mutants with disrupted interaction surfaces. It was validated that the self-polymerization of Spy0128 was prompted by the intermolecular cation-π interactions between Trp141 and Arg188 of the next monomer, which lowered the activation energy of the polymerization reaction. Furthermore, the polymerized protein nanoshackle shows great potential for the construction of functional detection reagents via incorporating functional moieties, including GFP and antigen−antibody like peptide binders, onto the N terminus of the self-assembly unit. Apart from disulfide bonds, the N-acyl rearrangement of Nterminal cysteine and C-terminal thioester has also been explored as a tool to guide covalent protein assembly. Main and co-workers developed a strategy in which designed repeat protein domains can be triggered to form superhelical filaments composed of a single protein chain through native chemical ligation (NCL) polymerization (Figure 84).307 The protein

Figure 82. (a) Design of TRAP tubes via intermolecular disulfide covalent linking of C69 and C50, respectively. (b) Protein nanotubes and (c) a bundle of the protein nanotubes. Scale bar is 100 nm. Adapted with permission from ref 305. Copyright 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

insights into the self-polymerizing mechanism of natural pili and fabricate protein nanofibrils in vitro, protein monomers based on a natural amino-acid sequence of Streptococcus pyogenes pilus subunit (Spy0128) were prepared. This artificial protein features an isopeptag at the N-terminus, while the isopeptag binding pocket at the C-terminus was protected by a cap peptide that is fixed by a disulfide bond (Figure 83a). Spy0128 can readily self-polymerize under redox conditions, which is attributed to the isopeptide bond formation facilitated by the disassociation of the disulfide bond (Figure 83b).

Figure 84. Covalent self-assembly of the designed repeat protein modules through the N-acyl rearrangement of N-terminal cysteine and C-terminal thioester. Topology map (a) and structures of CTPR3 (b) and CTPRa8 proteins (c). (d) Recombinant fusion protein. (e, f) CTPR3DS proteins with an N-terminal cysteine and C-terminal thioester. (g) Scheme depicting native chemical ligation (NCL) polymerization of protein monomers. Adapted with permission from ref 307. Copyright 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

building blocks were recombinantly engineered and folded into discrete conformationally rigid α-helical nonglobular domains. The specific head-to-tail self-assembly was driven by orthogonal native chemical ligation genetically encoded via intein chemistry. The protein building blocks were originally engineered to be monomeric through the genetic fusion of a cysteine residue at the N terminus and a thioesterified C terminus. The polymerization of the protein building block (CTPR) has been carried out by chemically linking the cysteine of one protein with the C-terminal thioester of another. The polymers consist of single protein chains that are helically folded and show a filamentous morphology with a length up to the micron scale. The individual filaments can further assemble into fibers, exhibiting heterogeneous branching and forming extensive networks. This work represents an efficient strategy

Figure 83. Schematic representation of (a) the design of the Spy0128 variant and (b) the polymerization process induced by isopeptide bond formation. Adapted with permission from ref 306. Copyright 2013 Nature Publishing Group. 13615


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that could be adapted for the synthesis of nano- or microscale protein assemblies. Although few examples of covalent protein assembly have been reported so far, this strategy has its own advantages: covalently cross-linked protein nanostructures displayed high stabilities compared with that by noncovalent methods; this issue is important for the development of functional materials. In an overall look, different design strategies offer their own advantages and disadvantages. As protein-assembly strategies continue to evolve and improve, ordered protein nanostructures should begin to find applications in broad areas of biomaterials, biosensing, and biocatalysis.

4. APPLICATIONS OF PROTEIN ASSEMBLIES Various kinds of protein assemblies with intricate structures and distinctive properties can be decorated with many useful components for a wide range of applications in the materials science and health science fields. The combination of the excellent characteristics of protein molecules (e.g., stable and definite structures, high recognition specificities, and efficient catalytic abilities) with those of functional nanoparticles, drug molecules, fluorescent dyes, and antibodies offers unique advantages over other self-assembled systems for the exploration of fundamental processes found in Nature or to create novel biomaterials with significantly improved performance over those achieved with pure molecules or particles. In this section, we will highlight recent examples of such functionalization with a focus on biosensors, biocatalysis, biomedical diagnosis and therapy, patterning metal nanoparticles, and other multifunctional materials.

Figure 85. (a) Self-assembled WPNFs that were functionalized with AuNPs and GOx for electrochemical applications; (b) self-assembly of Sup351−61-E2GFP−MPH fusion protein into nanowires for fluorescent-biosensor applications; (c) templated self-assembly of GFP− Ubx−QD nanowires to achieve a controllable QD distribution and an improved fiber extensibility for optical-biosensor applications. Adapted with permission from refs 313, 314, and 315. Copyright 2014 Royal Society of Chemistry, 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, and 2011 American Chemical Society, respectively.

4.1. Self-Assembled Protein Biosensors

The development of biosensing devices is an important application of protein assembly in medical-health-related monitoring and detection.308 Generally, biosensors consist of three components that perform roles that include probe-target recognition, signal transduction, and physical output.309 Protein building blocks with large surface-to-volume ratios and different kinds of reactive residues enable selective modification and bioconjugation of their assembly surfaces with signal molecules or nanoparticles (e.g., fluorescent molecules, QDs, and AuNPs) and biological recognition elements (e.g., enzymes, antibodies, and receptors) to construct nanoscale biosensors.310−312 These hybrid inorganic−protein-sensing composites are able to target (bio)chemical species in diverse environments and exhibit efficient electron transfer or fluorescence resonance energy transfer (FRET) for signal transduction. Some mature biological fibrils such as amyloid fibrils and ultrabithorax (Ubx) protein fibers have already demonstrated their advantages of structural stability and versatile surface chemistry for biosensor studies. For example, Sasso et al. developed an electrochemical biosensor based on the biotinylated whey protein nanofibrils (WPNFs) functionalized with streptavidin-coated GOx via the biotin−streptavidin interaction and subsequently thiolated by Traut’s reagent to immobilize gold nanoparticles onto their surfaces (Figure 85a).313 Typical cyclic voltammograms indicated highly efficient glucose detection of the multifunctionalized enzymatic biosensor that relies on the overall sensitivity and stability of protein assemblies. Using gene fusion technology, enzymes or fluorescent proteins can also be attached to amyloid proteins without impairing its activity for building an optical biosensor. Zhang

and co-workers reported the self-assembly of methyl parathion hydrolase (MPH), F64L/S65T/T203Y/L231H green fluorescent protein mutant (E2GFP), and amyloid-forming fragment Sup351−61 fusion protein fibrils as a nanowire fluorescent biosensor to detect pesticide methyl parathion (MP) by sensing the H+ ions released during MPH-catalyzed reactions (Figure 85b).314 Further studies confirmed that Sup351−61-E2GFP− MPH nanowires have the highest enzymatic activity, which was ∼3 times higher than that of the E2GFP−MPH sensors and ∼10.4% higher than that of the native free MPH. Due to the specific microenvironment of protein nanowires formed by selfassembly, this molecular biosensor exhibited a 104-fold increased detection sensitivity compared to E2GFP−MPH sensors. Similarly, Majithia et al. fabricated a hybrid protein− nanoparticle biomaterial that consists of GFP−Ubx fusion protein and luminescent CdSe−ZnS core−shell quantum dots (QDs) (Figure 85c).315 The hierarchical self-assembly of EGFP−Ubx offers a possibility of introducing the negatively or positively charged QDs into the protein fibers to form GFP− Ubx·QDs conjugates. SEM and confocal microscopy showed that smooth composite fibers can be constructed by the templated self-assembly of QDs onto EGFP−Ubx. In addition, the QD surface charge not only can affect QD distribution but also has a significant improvement on the extensibility of composite fibers, which can facilitate sensor design due to the enhanced FRET between photoluminescent QDs and fluorescent proteins. 13616


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4.2. Protein-Based Biocatalytic Nanomaterials

protein [(gp5bf)3]2-based nanotubes with the Ru and Re complexes were employed to construct an efficient photoreductase, which exhibited a higher photocatalytic CO2 reduction than that of the mixture of the monomeric forms. With the aid of computer modeling, Roelfes and co-workers precisely incorporated the CuII ligands into the hydrophobic pore of the self-assembled Lactococcal multidrug resistance Regulator (LmrR) dimer by cysteine conjugation (Figure 86b). Interestingly, enantioselective catalysis of the Diels−Alder reaction by the designed artificial metalloenzyme achieve up to 97% of yield, which may be attributed to the special chiral microenvironment formed by protein self-assembly.319 A similar strategy can be extended using a genetic engineering method. On the basis of theoretical simulations, Liu and coworkers designed and constructed a supramolecular artificial selenoenzyme in an auxotrophic system by engineering the tobacco mosaic virus (TMV) coat protein with a glutathione peroxidase (GPx)-like active site, including the catalytically essential residue selenocysteine (Sec) and substrate-binding residue Arg.320 Under different pH conditions, the genetically modified TMV monomers were able to self-assemble into welldefined nanodisks (pH = 7.0) or nanotubes (pH = 5.5) to form highly integrated nanoenzymes with surprising GPx activities (Figure 86c). Another strategy is the use of the confined spaces of natural/ non-natural protein assemblies such as ferritins, small heat shock proteins, and spherical viruses, which can form unique reaction environments and serve as nanoreactors for the catalysis of synthetic organic reactions via the encapsulation of organometallic complexes, inorganic nanoparticles, and enzymes inside their interior space. Abe et al. successfully synthesized poly(phenylacetylene) polymer within the Rh(norbornadiene)·apo-ferritin (Rh(nbd)·apo-Fr) cage in a controllable manner with a molecular weight distribution of (13.1 ± 1.5) × 103, which is a narrower distribution than that of the polymer produced by [Rh(nbd)Cl]2 ((63.7 ± 4) × 103) (Figure 87a).321 Engelkamp and co-workers investigated the enzymatic behavior of HRP at the single-molecule level inside the cowpea chlorotic mottle virus (CCMV) capsid (Figure 87b).322 The pH-dependent assembly/disassembly properties of CCMV are particularly advantageous to control the inclusion and release of enzyme molecules, and thereby each capsid can be manipulated to encapsulate only one HRP. Confocal

The design of sophisticated and complex biocatalysts to mimic cellular microcompartments can be realized by protein assembly when utilizing the well-ordered supramolecular architectures as scaffolds to introduce synthetic/natural cofactors, critical enzymatic components, or enzymes for important synthetic applications.316 Several technologies, such as computational methods, chemical conjugation, and biological engineering, were used to precisely design an inert and enzymelike active site into protein assemblies for high enzymatic activity and selectivity. This may provide opportunities not only to improve our understanding of complex cellular processes but also to create new catalytic reactions within the unique restricted nanospaces of protein assemblies.317 Site-specific modification of the catalytic moieties onto protein surfaces is the most widely used strategy to create new biocatalysts. Various metal complexes and organic ligands can be incorporated to achieve a high catalytic activity. For example, Ueno and co-workers reported that multimeric protein assembly provides a robust platform to covalently attach multiple catalytic sites (Figure 86a).318 Dual modifications of Lys and Cys on the surface of triple-stranded β-helix fusion

Figure 86. (a) Chemical modification of Ru(II) and Re(I) complexes on a fusion protein assembly to construct an efficient photoreductase. (b) Artificial metalloenzyme formed by grafting CuII ligands into the hydrophobic pore of dimeric LmrR for enantioselective catalysis. (c) TMV nanoenzymes with genetically engineered GPx-like active sites. Adapted with permission from refs 318, 319, and 320. Copyright 2011 Royal Society of Chemistry, 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, and 2012 American Chemical Society.

Figure 87. (a) Polymerization of phenylacetylene catalyzed by the Rh(nbd)·apo-Fr composite to achieve a narrower molecular weight distribution. (b) Single-molecule studies on the enzymatic behavior of HRP inside the CCMV capsid. Adapted with permission from refs 321 and 322. Copyright 2009 American Chemical Society and 2007 Nature Publishing Group, respectively. 13617


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fluorescence microscope characterization revealed that the CCMV capsid containing HRP exhibited higher localized fluorescence intensity in contrast to the mixture of nonencapsulated HRP and empty CCMV capsid when the fluorogenic substrate dihydrorhodamine 6G was used to examine their enzymatic activities. Although viruses and VLPs are considered as promising platforms for artificial enzyme development, the question of how to realize size control and multifunctionalization of nanoparticle assemblies is becoming a new challenge.323 In fact, protein nanoparticles are particularly advantageous for building size-controlled nanostructures due to their high degree of symmetry, availability for modification, and structural uniformity. As a proof-of-principle, Zhang and co-workers developed a size-controlled self-assembly strategy for the construction of multifunctional nanoparticle assemblies with tunable sizes by rational regulation of the number of selfassembling interaction sites on each ferritin nanoparticle (Figure 88a).324 The well-known biotin−STV interaction was adopted for the self-assembly of streptavidin-labeled horseradish peroxidase (STV−HRP) and autobiotinylated ferritin nanoparticles (ab-FNP) into a size-controlled enzyme nanocomposite (ENC). In this self-assembly system, one nanocomposite integrates a large number of enzyme molecules,

together with a streptavidin-coated surface, allowing for a drastic increase in the enzymatic signal when the streptavidin is bound to a biotinylated target molecule. A 10 000-fold increase in sensitivity, as compared with that of conventional enzymelinked immunosorbent assays (ELISA) methods, has been achieved in a cardiac troponin immunoassay. Therefore, it is feasible to create size-controlled ultrasensitive devices for a broad range of biomedical applications.324 Using recombinant DNA technology, several synergetic enzymes can also be displayed on the N/C-terminal of self-associating protein scaffolds in a manner of specific folding arrangements and diverse architectures that would be suitable for special industrial customization.325 In contrast to chemical cross-linking, enzyme production, purification, and immobilization can be accomplished in a single step during this process, which keeps the efficiency and yield of the active enzyme to its maximum extent.326 For example, Nagamune’s group used three distinct proliferating cell nuclear antigen (PCNA) protein subunits to form a self-assembled ring-shaped heterotrimer for the immobilization of a cytochrome P450 enzyme and its redox protein partners by genetic fusion. The resulting protein− enzyme complex was subsequently cross-linked with homodimeric phosphite dehydrogenase (PTDH) to yield a waterinsoluble gel, which displays monooxygenase activity and can achieve a synergistic electron transfer in the multicomponent system (Figure 88b). These studies may allow the circumvention of the current limitation that immobilized P450 enzyme requires auxiliary proteins for industrial usage.327 For the purpose of biomedical applications, protein assemblies with designated enzymatic functions need to be realized in living cells. Very recently, a significant contribution to this field was made by Tezcan and co-workers, who developed an artificial, in vivo active metallo-β-lactamase through ab initio design and Zn-mediated assembly of cytochrome (cyt) cb562 variants into tetrameric protein complexes (Zn8:AB34) (for details, see section 2.5). Due to the presence of the cyt cb562 variants with an N-terminal leader sequence, the designed supramolecular hydrolytic enzyme can be formed and accumulated in the Escherichia coli periplasm to endow the bacteria with ampicillin resistance. Moreover, saturation mutagenesis at a highly flexible loop near the tetrahedral zinc center of Zn8:AB34 enhances enzyme− substrate interactions and thereby allows in vivo selection and optimization for higher ampicillin hydrolysis activity (Figure 89a). This finding may represent a new insight into the mechanism of bacterial resistance to antibiotics via a rapid evolution of in vivo β-lactamase activity.179 In addition, large virus assemblies offer a powerful platform for in vivo assembly and encapsulation of the desired enzymes within their interiors for the construction of a new class of catalytically functional nanomaterials. Douglas and co-workers reported that the cagelike bacteriophage P22 capsid can be exploited as a container to encapsulate and protect an active hydrogenproducing and oxygen-tolerant [NiFe]-hydrogenase (Hyd) without altering the capsid morphology. The expression, maturation, and self-assembly of genetically fused P22-Hyd proteins in E. coli hosts led to the formation of a nanoparticle catalyst (Figure 89b), whose activity for proton reduction and hydrogen evolution was shown to increase 100-fold with respect to the purified, free enzyme, suggesting that the thermal stability and antiproteolytic effect of hydrogenase were greatly enhanced upon encapsulation.328

Figure 88. (a) Size-controlled self-assembly of ferritin nanoparticles into enzyme nanocomposites based on SA−HRP and abFNP for ultrasensitive immunoassays. Reprinted with permission from ref 324. Copyright 2015 American Chemical Society. (b) Immobilization of P450 enzyme and its redox protein partners on protein self-assemblies for synergistic catalysis. Adapted with permission from ref 327. Copyright 2015 Nature Publishing Group. 13618


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Figure 90. DNA-templated assembly of antibody (IgG) into very high density nanoarrays for potential immunodiagnostic applications. (i) AFM images of antigen-modified DNA arrays; (ii, iii) AFM images of antibody (IgG) arrays in solution and in air, respectively. Adapted with permission from ref 329. Copyright 2006 American Chemical Society.

N-terminal amino acids extensions (e.g., AVTS, FHKP, LAVG, or TS), and its C-terminus was fused to a polyhydroxybutyrate (PHB) synthase (PhaC), followed by a linker and foreign functional protein (e.g., maltose binding protein or IgG binding domains) to form large extensions (Figure 91). In the presence

Figure 89. (a) Investigation of in vivo β-lactamase activity of Zn8:AB34 that allows for in-lab evolution by point mutations. Reprinted with permission from ref 179. Copyright 2014 American Association for the Advancement of Science. (b) Stable nanoparticle catalyst with hydrogenase activity formed by in vivo self-assembly of P22-Hyd fusion proteins. Adapted with permission from ref 328. Copyright 2015 Nature Publishing Group.

4.3. Biomedical Diagnosis and Therapy

Protein assemblies are versatile scaffolds with definite structures and compositions, diverse properties, and multiple functional groups for conjugation with various biological molecules, such as antibodies, enzymes, and other species needed for diagnosis and therapy. For biomedical diagnostic applications, most of the effective strategies rely on the precise control of positions and orientations of these diagnostics. For example, Mao and coworkers developed periodic protein nanoarrays with high specificities and high affinities for diagnostic research.329 Antifluorescein antibodies were assembled into 2D tetragonal arrays templated along antigen-modified DNA lattices that contain nine single-strand DNA chains in well-ordered nanogrids with a uniform edge distance of ∼19 nm (Figure 90). To accomplish the self-organization of these components, two fluorescein molecules acting as antigens were fixed at the central loops of each cross motif of the tetragonal DNA, and an antibody (IgG) was immobilized onto the DNA arrays via strong antigen−antibody interactions. Because of the wellorientated antibody arrays assembled in solution with a very high density, they showed more advantages for in vitro and in vivo diagnostic applications than those immobilized on solid substrates. Another promising method for diagnostic assays is the in vivo assembly of protein particles for displaying the desired binding domains. The Rehm group demonstrated the power of this approach by employing an engineered green fluorescent protein (GFP) to self-assemble into protein particles in recombinant bacteria for mediating the display of various protein functionalities.330 GFP was modified with four different

Figure 91. Hybrid genes containing extended GFP, PhaC, and foreign functional proteins were expressed and then self-assembled into FP/ PHB hybrid particles for affinity-based diagnostic assays. TEM image of the isolated FP particles (i) and PHB/FP hybrid particles (ii). Adapted with permission from ref 330. Copyright 2013 American Chemical Society.

of the PhaC substrate, the insoluble fluorescent protein (FP)/ PHB hybrid particles were formed and mediated by N-terminal extensions to display specific binding proteins for applications in affinity-based diagnostic tests. Using a similar strategy, ultrasensitive diagnosis was achieved with natural protein particles that were engineered with specific antigenic epitopes to capture a disease marker. Lee’s group successfully constructed human ferritin heavy chain (hFTNH)/glutamate decarboxylase (GAD65)-based fusion protein nanoparticles with a significant enhancement of detection sensitivity.331 Owing to the formation of hFTN-H nanoparticle with 24 subunits, a number of epitopes were distributed outside the nanoparticles with a homogeneous and stable conforma13619


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cancer cells.337 In addition, bacteriophage MS2 VLPs are wellsuited for use in cell-specific drug delivery. Using genetic insertion or chemical conjugation, MS2 VLPs can be modified for a multivalent display of targeting ligands without impairing their ability to internalize siRNA- and RNA-conjugated cargos. For example, Ashley et al. reported that MS2 VLPs exhibited 104-fold higher selectivity toward human hepatocellular carcinoma (HCC) Hep3B cells than hepatocytes, endothelial cells, monocytes, and lymphocytes when they were modified with HCC-specific targeting peptide and loaded with imaging (e.g., QDs) and therapeutic agents (e.g., doxorubicin and ricin toxin A-chain), leading to selective cytotoxicity against cancer cells (Figure 93).338

tion, which offered multiple antigenic probes on each nanoparticle for the detection of GAD65-specific autoantibody (a disease marker of Type I diabetes), thereby resulting in 4−9 orders of magnitude greater sensitivity enhancement over that of conventional immunoassays. Park et al. developed a 3D diagnostic assay by combining hepatitis B virus (HBV) capsids with nickel nanohairs or porous membranes.332 To obtain dual affinity for troponin antibodies and nickel, the hexahistidine sequence (His6-Tag) was fused to the N-terminus of the HBV core protein and the P79A80 in the HBV loop segment was replaced by the tandem repeating of the B domains of staphylococcal protein A (SPAB) sequences (Figure 92). These

Figure 92. Enhanced sensitivity of 3D diagnostic assay can be realized by orienting the antibodies on the surface of self-assembled HBV nanoparticles. Adapted with permission from ref 332. Copyright 2009 Nature Publishing Group.

peptides and proteins were subsequently displayed on the surface HBV particles with high density and enabled control over the orientation of the antibodies for maximum capture of troponin markers, thus providing 6−7 orders of magnitude more sensitivity than conventional enzyme-linked immunosorbent assays in human serum samples. As far as biomedical therapy is concerned, protein assemblies with suitable size distributions and multiple functionalities are more effective in drug-loading applications,333 and precise delivery of the therapeutic agents into cell is greatly affected by their properties and functions (e.g., biocompatibility, biodegradability, specific targeting, and stimuli-responsiveness).334 Natural protein assemblies such as viruses, caged protein particles, and viral-like protein particles (VLPs) are attractive nanocarriers to covalently attach or encapsulate ligands and small molecules for drug delivery.335 Typically, cowpea mosaic virus (CPMV), cowpea chlorotic mottle virus (CCMV), canine parvovirus (CPV), and heat shock protein (HSP) cages were extensively studied by chemically or genetically engineering them with targeting ligands and drug molecules for specific tissue-targeting and therapy applications.336 Other successful examples such as zein nanoparticles that contain large amounts of hydrophobic amino acids are also a promising drug-delivery system to investigate the drug-release mechanism using different drugs and bioactive compounds such as ivermectin, coumarin, and 5-fluorouracil (5-FU). It was demonstrated that this delivery system can slowly release coumarin over 9 days in vitro and enabled multifunctional capabilities to treat breast

Figure 93. Self-assembled MS2 VLPs with targeting peptides can selectively deliver ricin toxin A-chain, QDs, and doxorubicin to Hep3B for cell-specific drug delivery. Confocal fluorescence microscopy images demonstrated that the modified MS2 VLPs (red) were internalized by Hep3B rather than hepatocytes. Adapted with permission from ref 338. Copyright 2011 American Chemical Society.

Antibody drug conjugation is also an important and viable way for targeted anticancer drug delivery, and several such conjugates have already received FDA approval for their clinical effects.339 Programmable self-assembly of antibodies offers a new class of platform to develop highly potent biopharmaceutical agents through combining the targeting capabilities of multivalent antibodies with the cytotoxicity of antitumor drugs. Wagner and co-workers proposed a methodology for the design of oligomeric antibody−drug conjugates through the chemical induction of protein assembly. For instance, a bismethotrexate 13620


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exploited for on-demand encapsulation and release of drug molecules in response to environmental changes. For instance, Aida and co-workers developed a smart tubular nanocarrier formed by Mg2+-mediated self-assembly of multiple barrelshaped chaperonin GroEL mutants.224 As a versatile scaffold, the internal and external surfaces of GroEL can be functionalized with a boronic acid (BA) derivative and an enzymatically cleavable ester-linked drug for cell penetration and drug encapsulation, respectively. In an in vivo cellular experiment, bioresponsive controlled drug release was triggered by an ATPfueled chemomechanical conformational change of GroEL that allowed ester−drug linkers to be accessible and subsequently cleaved by intracellular esterase (Figure 95a). In addition to ATP-responsive nanocarriers, the reversible opening and closing of surface pores of red clover necrotic mosaic virus (RCNMV) under different conditions (e.g., pH, buffer system, and concentration of divalent ions) are also able to self-regulate the electrostatically driven loading and release of anticancer

(bis-MTX) ligand with an additional single-stranded oligonucleotide was designed to induce the dimerization of dihydrofolate reductase (DHFR2)-antiCD3 scFv fusion proteins, in which oligonucleotide can be labeled with fluorescein/ therapeutic molecules or hybridized with another functionalized oligonucleotide to form DNA duplexes for carrying drugs.204 Flow cytometry analysis confirmed that this antibody− oligonucleotide conjugate has the capability to deliver small molecules and protein drugs into HPB-MLT cells in a concentration-dependent manner (Figure 94). Using a trivalent

Figure 94. Self-assembly of DHFR2-antiCD3 scFv fusion proteins via protein−ligand interactions or DNA complexation for targeted delivery of therapeutic molecules to HPB-MLT cells. Adapted with permission from ref 204. Copyright 2012 American Chemical Society.

bis-MTX ligand to attach drugs, higher-order antibody−drug conjugates were developed by intramolecular cyclization of DHFR2-antiCD3 fusion proteins for selective drug delivery.340 The chemically self-assembled antibody nanorings (CSANs) were observed to be rapidly internalized by HPB-MLT cells due to the specific interactions of scFv with CD3 cell-surface receptors and remained intact for several hours. However, adding a nontoxic DHFR inhibitor to the cells, efficient intracellular disassembly of these antibody nanorings and release of bis-MTX-drug can be achieved. This indicated that cell receptor targeting CSANs are promising drug carriers for selective delivery. The recent development of smart delivery systems based on stimuli-responsive protein assemblies has allowed for controlled drug release within cancer cells to further optimize the therapeutic efficacy. Many self-assembled protein nanostructures that are sensitive to specific endogenous stimuli and undergo reversible conformational transitions have been

Figure 95. (a) Mg2+-mediated self-assembly of GroEL mutants into tubular nanocarriers for ATP-responsive drug release. (b) RCNMV with pH- and ion-sensitive functions was used for controlled delivery of Dox. (c) pH-responsive drug release relies on the acid-dependent hydrolysis of the dynamic hydrazone bond between antitumor DOXOEMCH and E2 protein cage. Adapted with permission from refs 224, 341, and 342. Copyright 2013 Nature Publishing Group, 2014 and 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, respectively. 13621


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drug doxorubicin (Dox) (Figure 95b).341 One can take advantage of the pH- and ion-sensitive functions of RCNMV associated with the specific physiological microenvironment of human cancer cells for biomedical applications. Alternatively, the use of dynamic covalent bonds to chemically conjugate therapeutic agents to protein assemblies is a feasible route to achieve self-regulated drug delivery. Ren et al. designed a pHresponsive delivery system by coupling the (6-maleinimidocaproyl) hydrazone derivative of Dox (DOXO-EMCH) to the internal Cys381 of reengineered E2 (a component of pyruvate dehydrogenase) protein cage (D381C) through a Michael addition (Figure 95c). The acid-dependent hydrolysis of the hydrazine linker under the acidic cancer environment (pH = 5.0) caused 90% drug release from E2 scaffolds and thereby achieved improved cancer-therapy efficiency with reduced side effects.342 4.4. Other Functional Materials

Besides the above-mentioned applications, the symmetry, chemical/physical properties, and ease of structural manipulation and modification also make protein assemblies useful in many other nanotechnology applications, such as organizing metal nanoparticles into multidimensional patterns and creating new functional biomaterials with unprecedented properties. As an effective scaffold to display functional components, highly selective reactions on the surface of protein assemblies create a new possibility to template nanoparticles into welldefined arrays. These designed patterns with nanoscale precision could exhibit interesting optical and optoelectronic properties that depend upon their sizes, shapes, and interparticle distances. The related research has been conducted on AuNPs due to the well-studied surface chemistry and the stability of the gold−thiol bond. Paik and co-workers reported that dielectric amyloid protein-coated AuNPs were prepared by covalent attachment of the single cysteine residue of amyloid protein mutants onto the AuNP surface and then covered with the second layer of wild-type amyloid proteins for the control of nanoparticle assembly.343 Upon structural rearrangement induced by either hexane or a pH change, the designed AuNPs assembly units were aligned into an anisotropic chain structure during amyloid fibril formation and the average interparticle distances of AuNPs varied from 14.52 ± 5.4 nm (hexaneinduced chains) to 2.02 ± 0.38 nm (pH-induced chains) under different assembly conditions (Figure 96a). Photoconductivity studies revealed that pH-induced AuNP nanochains have a sensitive optic response at a normal electric field intensity due to the electron transfer favored by closely spaced AuNPs within dielectric amyloid fibril. This bottom-up nanofabrication method allows for the precise location and orientation control of noble-metal nanoparticles in a facile manner and therefore offers great potential for developing rapid-response optoelectric response systems near the surface plasmon resonance (SPR) frequency. Spatial arrangement of AuNPs can be realized by using the virus-based nanoparticles (VNPs) to provide topological control. On the basis of rational design and genetic engineering, viral capsid proteins were engineered with a cysteine moiety at the specific site for capturing AuNPs, and they subsequently underwent VNP-templated assembly into deliberately designed patterns. Zhang and co-workers employed this strategy to construct discrete 3D hybrid Au/QD nanoarchitectures. Simian virus 40 (SV40) capsid with an icosahedral symmetry and a A74C mutation was utilized as a robust scaffold to encapsulate

Figure 96. Controlled spatial arrangement of AuNPs into precisely defined arrays by amyloid proteins (a) and virus-based nanoparticles (b). Adapted with permission from refs 343 and 345. Copyright 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, and 2011 American Chemical Society, respectively.

3-mercaptopropionic acid (MPA)-coated QDs and to deposit citrate-capped AuNPs via molecular self-assembly and strong thiol/gold affinity, respectively.344 The formation of AuNP clusters on SV40 surfaces can be controlled by altering the ratio of AuNPs to QD-VNPs to obtain narrow distributions, which resulted in a red-shift of the SPR. In addition, the dramatic influence of AuNP arrangements on the fluorescence intensity of QDs also confirmed that plasmon coupling occurred. It has been suggested that VNP-templated assembly method allows the creation of AuNP arrays with controllable number, interparticle distance, and morphology through highly selective surface functionalization. This strategy was later optimized by the same group to develop Janus-like Au/QD-VNP hybrid protein nanostructures.345 An improved protein building block (5hcVP1) was established by incorporating a tetrahistidine tag after the surface-exposed histidine 139 of SV40 A74C variant for purification purposes. Coassembly of wild-type SV40 subunit (wtVP1) and 5hcVP1 at a molar ratio of 11:1 led to the formation of monofunctionalized Au/QD-VNP nanostructures when QDs and AuNPs were introduced into the assembly process (Figure 96b). Nickel affinity chromatography makes it possible to achieve an excellent separation of the heterogeneous protein assemblies with one QD and one AuNP from the assembling mixture, revealing that protein assembly is a versatile platform for organizing and purifying nanoparticles. Taking advantage of the chemical versatility of protein assemblies, multiple approaches have been explored to expand the biological functions and properties of protein-based materials. Seki and co-workers reported the use of a singleparticle nanofabrication technique (SPNT) to develop enzymedegradable and size-controlled protein nanowires.346 Highenergy charged particle irradiation enables human serum 13622


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attractive properties, and versatile functions. On the basis of creative biological and chemical technologies, templated and nontemplated self-assembly protocols were described. We also summarized the main strategies such as computational design, symmetry design, electrostatic induction, and host−guest interactions that guide the protein−protein interactions for controlling the order and orientation of protein selforganization processes. By rational design and site-specific modification of the protein interface, the direct manipulation of proteins at the molecular level to form large-scale, well-ordered superstructures ranging from 1D polymeric, fibrous, and nanotubular nanostructures to 3D cross-linked, micellar, and spherical morphologies can be achieved through external noncovalent driving forces. These design strategies were highlighted, and we subsequently provided a critical discussion of their advantages and potential applications. Protein assembly, while young, has become an exciting field for multidisciplinary research. In contrast to other self-assembly systems, it offers unique advantages not only for understanding the natural assembly process but also for opening many prospects to address the materials and health issues. First, a limitless variety of protein monomers or oligomers offer a rich source of complicated building blocks for programmable selfassembling and coassembling into intriguing biomaterials analogous to those found in Nature, which is impossible to accomplish by traditional chemical molecules. Next, an in-depth understanding of the underlying mechanism of protein aggregation provides a profound insight into the synergistic effect of protein−protein interactions whose strength and directionality can be modulated to explore novel protein superstructures in a predictable way. It will be envisioned to aid both in clarifying Nature’s bottom-up fabrication processes and in developing new treatments for protein-aggregation-related disorders. Finally, proteins are known to exhibit atomic resolution, and their assemblies are capable of selective modification with various functional groups, molecules, and nanoparticles to achieve desirable properties for many promising applications. Despite impressive progress in recent years, in order to proceed from in vitro protein self-assembly to in vivo protein self-assembly, further substantial efforts are still required to explore the mechanism and correlation of different protein associations. This is particularly needed in order to reveal the general behavior of multiple noncovalent dominated interactions between extensive, heterogeneous protein−protein interfaces. It is expected that a set of principles or rules could be established according to the collected theoretical and experimental data for de novo design of proteins into arbitrary symmetric architectures by utilizing the newly engineered protein interfaces rather than the existing recognition interfaces or well-known self-associating motifs. In addition, the flexible combination of two or more biotechnological and chemical strategies could customize the complexity of the next generation of protein assemblies and ultimately create exciting novel biomaterials that extend beyond biomimicry. With respect to biomedical applications, both the sophisticated structural features of protein assemblies and the intrinsic properties of protein units should be considered to achieve an optimal design strategy for protein functionalization. Furthermore, the stability, reparative ability, pharmacokinetics, adverse host reactions, toxic effects, and biodegradation of proteinbased biomaterials need to be systematically evaluated in vivo. The goal of generating highly ordered protein assemblies with

albumin (HSA) to undergo an efficient cross-linking reaction along the ion track trajectories to produce 1D protein nanostructures, which are controllable and can be transformed from nanodots to nanowires with an ultrahigh aspect ratio of >1 000 by adjusting the thickness of the film (Figure 97a). The

Figure 97. Fabrication of enzyme-degradable and size-controlled protein nanowires (a) and free-standing protein films (b) by SPNT and a scalable self-assembly approach, respectively. Adapted with permission from refs 346 and 347. Copyright 2014 and 2010 Nature Publishing Group, respectively.

HSA nanowires were shown to be susceptible to tryptic degradation by cleaving the peptide bonds at the C-terminal side of lysine and arginine residues. As a versatile platform, the surface of the HSA nanowires was easily modified with the biotinyl group for specific binding of Alexa Fluor 488conjugated streptavidin and treptavidin-conjugated HRP, showing the flexibility and ability to fabricate various biomaterials with diverse biological functions. Another novel approach to realize sophisticated functional materials was described by Welland and co-workers, who used a scalable selfassembly approach to construct free-standing protein films.347 The method involved two steps. The first step involved the selfassembly of amyloid protein monomers into highly rigid nanofibrils, and the second step was to cast the nanofibrils into macroscopic thin films, where a plasticizer was added as a lubricant to increase their elongation and processability by reducing the interfibril friction (Figure 97b). Three-point flexural test has determined the mechanical properties of artificial biofilms that reach the level of natural materials such as keratin and collagen with a Young’s modulus of up to 5−7 GPa. Because unstructured molecules (e.g., fluorophores) can be well-arranged within the films, the self-assembling protein scaffolds could offer great opportunities to combine the desirable properties (e.g., fluorescence) of introduced components with the characteristics of nanofilms for developing new multifunctional materials.

5. CONCLUSION AND OUTLOOK In this Review, we presented an exhaustive overview of different supramolecular routes toward the construction of selfassembled protein nanostructures with diverse architectures, 13623


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advanced functions and wide-ranging applications in a reliable, controlled, and reproducible way will constitute an important future challenge, which requires an intimate collaboration between scientists in various disciplines such as nanoscience, materials science, and chemical biology as well as structural and synthetic biology to support the rapid development of this highly interdisciplinary research field.

systems, biomolecule-based supramolecular self-assembly, and bionanomaterials.

ACKNOWLEDGMENTS We acknowledge the financial support from the National Natural Science Foundation of China (nos. 21234004, 21420102007, 21574056, 91527302, 21474038, and 21004028), the Chang Jiang Scholars Program of China, and the Science Development Program of Jilin Province (nos. 20140101047JC and 20160520005JH).

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Notes

REFERENCES

The authors declare no competing financial interest.

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Biographies Quan Luo received his Ph.D. in Physical Chemistry from Jilin University, China, under the supervision of Prof. Zesheng Li in 2009. Subsequently, he became an assistant professor at the State Key Laboratory of Supramolecular Structure and Materials of the same university and was promoted to become an associate professor in 2012. He is currently a visiting scholar at the Department of Chemistry, University of Minnesota, U.S.A. His main research interests include molecular design of artificial enzymes and exploitation of bioactive materials via protein self-assembly. Chunxi Hou has been a postdoctoral fellow from 2010 to 2013 under the supervision of Prof. Junqiu Liu at the State Key Laboratory of Supramolecular Structure and materials, Jilin University, China. He received his Ph.D. from the College of Biological and Agricultural Engineering, Jilin University, in 2009 under the supervision of Prof. Shoujing Zhao. He is now an assistant professor in Prof. Junqiu Liu’s Laboratory at the State Key Laboratory of Supramolecular Structure and Materials. His main research interests focus on the construction of supramolecular protein assemblies by biotechnology and chemical synthesis and their use for biomedical applications. Yushi Bai received his B.Sc. degree in Chemistry from Jilin University, China, in 2011. Currently he is a Ph.D. student under the supervision of Prof. Junqiu Liu at the State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, China. His recent research has focused on the design and construction of sophisticated protein assemblies. Ruibing Wang received his B.S. degree in chemistry from Jilin University, China, in 2002 and his Ph.D. degree in organic chemistry from Queen’s University, Canada, in 2007. Shortly after graduation, he moved to the Steacie Institute for Molecular Sciences at the National Research Council, Ottawa, Canada, as a research associate for about 2 years. From 2009 to 2014, he worked as a senior scientist at BTG International, Ottawa, Canada, for 5 years. Since late 2014, he has joined the Institute of Chinese Medical Sciences at University of Macau, Macau SAR, as an assistant professor of biomedical sciences. His research interests include, but are not limited to, supramolecular chemistry for pharmaceutical sciences, nanobiotechnology, and interventional medicine. Junqiu Liu received his Ph.D. from the State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, China, in 1999 under the supervision of Professor Jiacong Shen. Following his doctoral studies, he was a Humboldt Fellow and a Postdoctoral Fellow with Professor Günter Wulff at the Institute of Organic and Macromolecular Chemistry, Heinrich-Heine University, Germany. In 2003 he joined the faculty of the State Key Laboratory of Supramolecular Structure and Materials at Jilin University as a full professor of chemistry. His main research interests include biomimetic 13624


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