Engineering a Self-Assembled Protein Cage for Targeted Dual Functionalization.

Self-assembled protein cages are attractive scaffolds for organizing various proteins of interest (POIs) toward applications in synthetic biology and medical science. However, specifically attaching multiple POIs to a single protein cage remains challenging, resulting in diversity among the functionalized particles. Here, we present the engineering of a self-assembled protein cage, DTMi3ST, capable of independently recruiting two different POIs using SpyCatcher (SC)/SpyTag (ST) and DogCatcher (DC)/DogTag (DT) chemistries, thereby reducing variability between assemblies. Using fluorescent proteins as models, we demonstrate controlled targeting of two different POIs onto DTMi3ST protein cages both in vitro and inside living cells. Furthermore, dual functionalization of the DTMi3ST protein cage with a membrane-targeting peptide and ß-galactosidase resulted in the construction of membrane-bound enzyme assemblies in Escherichia coli, leading to a 69.6% enhancement in substrate utilization across the membrane. This versatile protein cage platform provides dual functional nanotools for biological and biomedical applications.

Magnetically Induced Thermal Effects on Tobacco Mosaic Virus-Based Nanocomposites for a Programmed Disassembly of Protein Cages.

Protein cages are promising tools for the controlled delivery of therapeutics and imaging agents when endowed with programmable disassembly strategies. Here, we produced hybrid nanocomposites made of tobacco mosaic virus (TMV) and magnetic iron oxide nanoparticles (IONPs), designed to disrupt the viral protein cages using magnetically induced release of heat. We studied the effects of this magnetic hyperthermia on the programmable viral protein capsid disassembly using (1) elongated nanocomposites of TMV coated heterogeneously with magnetic iron oxide nanoparticles (TMV@IONPs) and (2) spherical nanocomposites of polystyrene (PS) on which we deposited presynthesized IONPs and TMV via layer-by-layer self-assembly (PS@IONPs/TMV). Notably, we found that the extent of the disassembly of the protein cages is contingent upon the specific absorption rate (SAR) of the magnetic nanoparticles, that is, the heating efficiency, and the relative position of the protein cage within the nanocomposite concerning the heating sources. This implies that the spatial arrangement of components within the hybrid nanostructure has a significant impact on the disassembly process. Understanding and optimizing this relationship will contribute to the critical spatiotemporal control for targeted drug and gene delivery using protein cages.

Iron mobilization from intact ferritin: effect of differential redox activity of quinone derivatives with NADH/O2 and in situ-generated ROS.

Ferritins are multimeric nanocage proteins that sequester/concentrate excess of free iron and catalytically synthesize a hydrated ferric oxyhydroxide bio-mineral. Besides functioning as the primary intracellular iron storehouses, these supramolecular assemblies also oversee the controlled release of iron to meet physiologic demands. By virtue of the reducing nature of the cytosol, reductive dissolution of ferritin-iron bio-mineral by physiologic reducing agents might be a probable pathway operating in vivo. Herein, to explore this reductive iron-release pathway, a series of quinone analogs differing in size, position/nature of substituents and redox potentials were employed to relay electrons from physiologic reducing agent, NADH, to the ferritin core. Quinones are well known natural electron/proton mediators capable of facilitating both 1/2 electron transfer processes and have been implicated in iron/nutrient acquisition in plants and energy transduction. Our findings on the structure-reactivity of quinone mediators highlight that iron release from ferritin is dictated by electron-relay capability (dependent on E1/2 values) of quinones, their molecular structure (i.e., the presence of iron-chelation sites and the propensity for H-bonding) and the type/amount of reactive oxygen species (ROS) they generate in situ. Juglone/Plumbagin released maximum iron due to their intermediate E1/2 values, presence of iron chelation sites, the ability to inhibit in situ generation of H2O2 and form intramolecular H-bonding (possibly promotes semiquinone formation). This study may strengthen our understanding of the ferritin-iron-release process and their significance in bioenergetics/O2-based cellular metabolism/toxicity while providing insights on microbial/plant iron acquisition and the dynamic host-pathogen interactions.

Point mutation in a virus-like capsid drives symmetry reduction to form tetrahedral cages.

Protein capsids are a widespread form of compartmentalization in nature. Icosahedral symmetry is ubiquitous in capsids derived from spherical viruses, as this geometry maximizes the internal volume that can be enclosed within. Despite the strong preference for icosahedral symmetry, we show that simple point mutations in a virus-like capsid can drive the assembly of unique symmetry-reduced structures. Starting with the encapsulin from Myxococcus xanthus, a 180-mer bacterial capsid that adopts the well-studied viral HK97 fold, we use mass photometry and native charge detection mass spectrometry to identify a triple histidine point mutant that forms smaller dimorphic assemblies. Using cryoelectron microscopy, we determine the structures of a precedented 60-mer icosahedral assembly and an unexpected 36-mer tetrahedron that features significant geometric rearrangements around a new interaction surface between capsid protomers. We subsequently find that the tetrahedral assembly can be generated by triple-point mutation to various amino acids and that even a single histidine point mutation is sufficient to form tetrahedra. These findings represent a unique example of tetrahedral geometry when surveying all characterized encapsulins, HK97-like capsids, or indeed any virus-derived capsids reported in the Protein Data Bank, revealing the surprising plasticity of capsid self-assembly that can be accessed through minimal changes in the protein sequence.

Virus-like particles derived from bacteriophage MS2 as antigen scaffolds and RNA protective shells.

The versatile potential of bacteriophage MS2-derived virus-like particles (VLPs) in medical biotechnology has been extensively studied during the last 30 years. Since the first reports showing that MS2 VLPs can be produced at high yield and relatively easily engineered, numerous applications have been proposed. Particular effort has been spent in developing MS2 VLPs as protective capsules and delivery platforms for diverse molecules, such as chemical compounds, proteins and nucleic acids. Among these, two are particularly noteworthy: as scaffolds displaying heterologous epitopes for vaccine development and as capsids for encapsulation of foreign RNA. In this review, we summarize the progress in developing MS2 VLPs for these two areas.

Hollow, nanosized protein particles have many potential uses. If they can be appropriately engineered, they may for example be able to carry therapeutic cargoes to diseased cells or be used as a vaccine where appropriate antigens are mounted on their external surface. Many viruses offer a ready-made protein particle, the capsid, which can be made hollow by exclusion of the viral genetic material. MS2 is a virus that targets bacteria ­ a bacteriophage ­ which is well characterized and has been developed over many years for a number of applications. It has particular promise for development as a vaccine and for RNA delivery, both of which are reviewed here.

Designed fluorescent protein cages as fiducial markers for targeted cell imaging.

Understanding how proteins function within their cellular environments is essential for cellular biology and biomedical research. However, current imaging techniques exhibit limitations, particularly in the study of small complexes and individual proteins within cells. Previously, protein cages have been employed as imaging scaffolds to study purified small proteins using cryo-electron microscopy (cryo-EM). Here we demonstrate an approach to deliver designed protein cages - endowed with fluorescence and targeted binding properties - into cells, thereby serving as fiducial markers for cellular imaging. We used protein cages with anti-GFP DARPin domains to target a mitochondrial protein (MFN1) expressed in mammalian cells, which was genetically fused to GFP. We demonstrate that the protein cages can penetrate cells, are directed to specific subcellular locations, and are detectable with confocal microscopy. This innovation represents a milestone in developing tools for in-depth cellular exploration, especially in conjunction with methods such as cryo-correlative light and electron microscopy (cryo-CLEM).

Virus-like Particles Armored by an Endoskeleton.

Many virus-like particles (VLPs) have good chemical, thermal, and mechanical stabilities compared to those of other biologics. However, their stability needs to be improved for the commercialization and use in translation of VLP-based materials. We developed an endoskeleton-armored strategy for enhancing VLP stability. Specifically, the VLPs of physalis mottle virus (PhMV) and Qß were used to demonstrate this concept. We built an internal polymer "backbone" using a maleimide-PEG15-maleimide cross-linker to covalently interlink viral coat proteins inside the capsid cavity, while the native VLPs are held together by only noncovalent bonding between subunits. Endoskeleton-armored VLPs exhibited significantly improved thermal stability (95 °C for 15 min), increased resistance to denaturants (i.e., surfactants, pHs, chemical denaturants, and organic solvents), and enhanced mechanical performance. Single-molecule force spectroscopy demonstrated a 6-fold increase in rupture distance and a 1.9-fold increase in rupture force of endoskeleton-armored PhMV. Overall, this endoskeleton-armored strategy provides more opportunities for the development and applications of materials.

Displaying a Protein Cage on a Protein Crystal by In-Cell Crystal Engineering.

The development of solid biomaterials has rapidly progressed in recent years in applications in bionanotechnology. The immobilization of proteins, such as enzymes, within protein crystals is being used to develop solid catalysts and functionalized materials. However, an efficient method for encapsulating protein assemblies has not yet been established. This work presents a novel approach to displaying protein cages onto a crystalline protein scaffold using in-cell protein crystal engineering. The polyhedra crystal (PhC) scaffold, which displays a ferritin cage, was produced by coexpression of polyhedrin monomer (PhM) and H1-ferritin (H1-Fr) monomer in Escherichia coli. The H1-tag is derived from the H1-helix of PhM. Our technique represents a unique strategy for immobilizing protein assemblies onto in-cell protein crystals and is expected to contribute to various applications in bionanotechnology.

Glucose oxidase virus-based nanoreactors for smart breast cancer therapy.

BACKGROUND: Breast cancer is the most common malignant tumor disease and the leading cause of female mortality. The evolution of nanomaterials science opens the opportunity to improve traditional cancer therapies, enhancing therapy efficiency and reducing side effects. METHODS AND MAJOR RESULTS: Herein, protein cages conceived as enzymatic nanoreactors were designed and produced by using virus-like nanoparticles (VLPs) from Brome mosaic virus (BMV) and containing the catalytic activity of glucose oxidase (GOx) enzyme. The GOx enzyme was encapsulated into the BMV capsid (VLP-GOx), and the resulting enzymatic nanoreactors were coated with human serum albumin (VLP-GOx@HSA) for breast tumor cell targeting. The effect of the synthesized GOx nanoreactors on breast tumor cell lines was studied in vitro. Both nanoreactor preparations VLP-GOx and VLP-GOx@HSA showed to be highly cytotoxic for breast tumor cell cultures. Cytotoxicity for human embryonic kidney cells was also found. The monitoring of nanoreactor treatment on triple-negative breast cancer cells showed an evident production of oxygen by the catalase antioxidant enzyme induced by the high production of hydrogen peroxide from GOx activity. CONCLUSIONS AND IMPLICATIONS: The nanoreactors containing GOx activity are entirely suitable for cytotoxicity generation in tumor cells. The HSA functionalization of the VLP-GOx nanoreactors, a strategy designed for selective cancer targeting, showed no improvement in the cytotoxic effect. The GOx containing enzymatic nanoreactors seems to be an interesting alternative to improve the current cancer therapy. In vivo studies are ongoing to reinforce the effectiveness of this treatment strategy.

Artificial Protein Cages Assembled via Gold Coordination.

Artificial protein cages made from multiple copies of a single protein can be produced such that they only assemble upon addition of a metal ion. Consequently, the ability to remove the metal ion triggers protein-cage disassembly. Controlling assembly and disassembly has many potential uses including cargo loading/unloading and hence drug delivery. TRAP-cage is an example of such a protein cage which assembles due to linear coordination bond formation with Au(I) which acts to bridge constituent proteins. Here we describe the method for production and purification of TRAP-cage.

Protein Cages and Nanostructures Constructed from Protein Nanobuilding Blocks.

Protein cages and nanostructures are promising biocompatible medical materials, such as vaccines and drug carriers. Recent advances in designed protein nanocages and nanostructures have opened up cutting-edge applications in the fields of synthetic biology and biopharmaceuticals. A simple approach for constructing self-assembling protein nanocages and nanostructures is the design of a fusion protein composed of two different proteins forming symmetric oligomers. In this chapter, we describe the design and methods of protein nanobuilding blocks (PN-Blocks) using a dimeric de novo protein WA20 to construct self-assembling protein cages and nanostructures. A protein nanobuilding block (PN-Block), WA20-foldon, was developed by fusing an intermolecularly folded dimeric de novo protein WA20 and a trimeric foldon domain from bacteriophage T4 fibritin. The WA20-foldon self-assembled into several oligomeric nanoarchitectures in multiples of 6-mer. De novo extender protein nanobuilding blocks (ePN-Blocks) were also developed by fusing tandemly two WA20 with various linkers, to construct self-assembling cyclized and extended chain-like nanostructures. These PN-Blocks would be useful for the construction of self-assembling protein cages and nanostructures and their potential applications in the future.

A Generalized Method for Metal Fixation in Horse Spleen L-Ferritin Cage.

The naturally occurring iron storage protein, ferritin, has been recognized as an important template for preparing inorganic nanomaterials by fixation of metal ions and metal complexes into the cage. Such ferritin-based biomaterials find applications in various fields like bioimaging, drug delivery, catalysis, and biotechnology. The unique structural features with exceptional stability at high temperature up to ca. 100 °C and a wide pH range of 2-11 enable to design the ferritin cage for such interesting applications. Infiltration of metals into ferritin is one of the key steps for preparing ferritin-based inorganic bionanomaterials. Metal-immobilized ferritin cage can be directly utilized for applications or act as a precursor for synthesizing monodisperse and water-soluble nanoparticles. Considering this, herein, we have described a general protocol on how to immobilize metal into a ferritin cage and crystallize the metal composite for structure determination.

Construction Protocol of Drug-Protein Cage Complexes for Drug Delivery System.

Ferritin is one of the most promising drug delivery system (DDS) carriers because of its uniform nanosize, biodistribution, efficient cellular uptake, and biocompatibility. Conventionally, a disassembly/reassembly method that requires pH change has been used for the encapsulation of molecules in ferritin protein nanocages. Recently, a one-step method in which a complex of ferritin and a targeted drug was obtained by incubating the mixture at an appropriate pH, was established. Here, we describe two types of protocols, the conventional disassembly/reassembly method, and the novel one-step method for the construction of a ferritin-encapsulated drug using doxorubicin as an example molecule.

Crystalline Biohybrid Materials Based on Protein Cages.

Highly ordered superstructures of nanomaterials can be synthesized using protein cages as templates for the assembly of inorganic nanoparticles. Here, we describe in detail the creation of these biohybrid materials. The approach involves computational redesign of ferritin cages, followed by recombinant protein production and purification of the new variants. Metal oxide nanoparticles are synthesized inside the surface-charged variants. The composites are assembled using protein crystallization to yield highly ordered superlattices, which are characterized, for example, with small angle X-ray scattering. This protocol provides a detailed and comprehensive account on our newly established strategy for the synthesis of crystalline biohybrid materials.

Optically Controlled Construction of Three-Dimensional Protein Arrays.

Protein crystallization is an important tool for structural biology and nanostructure preparation. Here, we report on kinetic pathway-dependent protein crystals that are controlled by light. Photo-responsive crystallites are obtained by complexing the model proteins with cationic azobenzene dyes. The crystalline state is readily switched to a dispersed phase under ultraviolet light and restored by subsequent visible-light illumination. The switching can be reversibly repeated for multiple cycles without noticeable structure deterioration. Importantly, the photo-treatment not only significantly increases the crystallinity, but creates crystallites at conditions where no ordered lattices are observed upon directly mixing the components. Further control over the azobenzene isomerization kinetics produces protein single crystals of up to ≈50 µm. This approach offers an intriguing method to fabricate metamaterials and study optically controlled crystallization.

Elucidating Conformational Dynamics and Thermostability of Designed Aromatic Clusters by Using Protein Cages.

Multiple aromatic residues assemble to form higher ordered structures known as "aromatic clusters" in proteins and play essential roles in biological systems. However, the stabilization mechanism and dynamic behavior of aromatic clusters remain unclear. This study describes designed aromatic interactions confined within a protein cage to reveal how aromatic clusters affect protein stability. The crystal structures and calorimetric measurements indicate that the formation of inter-subunit phenylalanine clusters enhance the interhelix interactions and increase the melting temperature. Theoretical calculations suggest that this is caused by the transformation of the T-shaped geometry into π-π stacking at high temperatures, and the hydration entropic gain. Thus, the isolated nanoenvironment in a protein cage allows reconstruction and detailed analysis of multiple clustering residues for elucidating the mechanisms of various biomolecular interactions in nature which can be applied to design of bionanomaterials.

Tunable In Vivo Colocalization of Enzymes within P22 Capsid-Based Nanoreactors.

Virus-like particles (VLPs) derived from bacteriophage P22 have been explored as biomimetic catalytic compartments. In vivo colocalization of enzymes within P22 VLPs uses sequential fusion to the scaffold protein, resulting in equimolar concentrations of enzyme monomers. However, control over enzyme stoichiometry, which has been shown to influence pathway flux, is key to realizing the full potential of P22 VLPs as artificial metabolons. We present a tunable strategy for stoichiometric control over in vivo co-encapsulation of P22 cargo proteins, verified for fluorescent protein cargo by Förster resonance energy transfer. This was then applied to a two-enzyme reaction cascade. l-homoalanine, an unnatural amino acid and chiral precursor to several drugs, can be synthesized from the readily available l-threonine by the sequential activity of threonine dehydratase and glutamate dehydrogenase. We found that the loading density of both enzymes influences their activity, with higher activity found at lower loading density implying an impact of molecular crowding on enzyme activity. Conversely, increasing overall loading density by increasing the amount of threonine dehydratase can increase activity from the rate-limiting glutamate dehydrogenase. This work demonstrates the in vivo colocalization of multiple heterologous cargo proteins in a P22-based nanoreactor and shows that controlled stoichiometry of individual enzymes in an enzymatic cascade is required for the optimal design of nanoscale biocatalytic compartments.

Bacteriophage P22 Capsid as a Pluripotent Nanotechnology Tool.

The Salmonella enterica bacteriophage P22 is one of the most promising models for the development of virus-like particle (VLP) nanocages. It possesses an icosahedral T = 7 capsid, assembled by the combination of two structural proteins: the coat protein (gp5) and the scaffold protein (gp8). The P22 capsid has the remarkable capability of undergoing structural transition into three morphologies with differing diameters and wall-pore sizes. These varied morphologies can be explored for the design of nanoplatforms, such as for the development of cargo internalization strategies. The capsid proteic nature allows for the extensive modification of its structure, enabling the addition of non-native structures to alter the VLP properties or confer them to diverse ends. Various molecules were added to the P22 VLP through genetic, chemical, and other means to both the capsid and the scaffold protein, permitting the encapsulation or the presentation of cargo. This allows the particle to be exploited for numerous purposes-for example, as a nanocarrier, nanoreactor, and vaccine model, among other applications. Therefore, the present review intends to give an overview of the literature on this amazing particle.

Dissecting the general mechanisms of protein cage self-assembly by coarse-grained simulations.

The development of artificial protein cages has recently gained massive attention due to their promising application prospect as novel delivery vehicles for therapeutics. These nanoparticles are formed through a process called self-assembly, in which individual subunits spontaneously arrange into highly ordered patterns via non-covalent but specific interactions. Therefore, the first step toward the design of novel engineered protein cages is to understand the general mechanisms of their self-assembling dynamics. Here we have developed a new computational method to tackle this problem. Our method is based on a coarse-grained model and a diffusion-reaction simulation algorithm. Using a tetrahedral cage as test model, we showed that self-assembly of protein cage requires of a seeding process in which specific configurations of kinetic intermediate states are identified. We further found that there is a critical concentration to trigger self-assembly of protein cages. This critical concentration allows that cages can only be successfully assembled under a persistently high concentration. Additionally, phase diagram of self-assembly has been constructed by systematically testing the model across a wide range of binding parameters. Finally, our simulations demonstrated the importance of protein's structural flexibility in regulating the dynamics of cage assembly. In summary, this study throws lights on the general principles underlying self-assembly of large cage-like protein complexes and thus provides insights to design new nanomaterials.

Assembly of Protein Cages for Drug Delivery.

Nanoparticles (NPs) have been widely used as target delivery vehicles for therapeutic goods; however, compared with inorganic and organic nanomaterials, protein nanomaterials have better biocompatibility and can self-assemble into highly ordered cage-like structures, which are more favorable for applications in targeted drug delivery. In this review, we concentrate on the typical protein cage nanoparticles drugs encapsulation processes, such as drug fusion expression, diffusion, electrostatic contact, covalent binding, and protein cage disassembly/recombination. The usage of protein cage nanoparticles in biomedicine is also briefly discussed. These materials can be utilized to transport small molecules, peptides, siRNA, and other medications for anti-tumor, contrast, etc.