Engineering the HK97 virus-like particle as a nanoplatform for biotechnology applications.

The research described here looks at the development of virus-like particles (VLPs) derived from bacteriophage HK97 as versatile scaffolds for bionanomaterials construction. Based on molecular models, the Prohead I HK97 VLP was engineered to allow attachment of small molecules to the interior by introducing a reactive cysteine into the genetic sequence of the HK97 GP5 protein that self assembles to form the VLP structure. In addition, methods for entrapping large protein macromolecules were evaluated and found to produce high encapsulation numbers of green fluorescent proteins (GFP) in the internal space of the HK97 VLP. A method for modular modification of the external surface was engineered by constructing a plasmid allowing the addition of peptide sequences to the C-terminus of the GP5 protein, which was validated by appending the sortase recognition peptide sequence, LPETG, to the C-terminus of GP5 and showing the attachment of a polyglycine-GFP to the HK97 VLP through sortase mediated ligation. To demonstrate the potential for advanced applications, an HK97 VLP covalently labeled on the interior surface with fluorescein and containing an externally displayed integrin binding peptide sequence (RGD) was evaluated and found to be preferentially localized at C2C12 cells relative to the HK97 VLP lacking the RGD peptide. Together, these results support the potential of the HK97 VLP as a versatile nanoparticle platform that can be modified internally and externally in a modular fashion for the purpose of programming the VLP for desired applications.

Engineering DNA recognition and allosteric response properties of TetR family proteins by using a module-swapping strategy.

The development of synthetic biological systems requires modular biomolecular components to flexibly alter response pathways. In previous studies, we have established a module-swapping design principle to engineer allosteric response and DNA recognition properties among regulators in the LacI family, in which the engineered regulators served as effective components for implementing new cellular behavior. Here we introduced this protein engineering strategy to two regulators in the TetR family: TetR (UniProt Accession ID: P04483) and MphR (Q9EVJ6). The TetR DNA-binding module and the MphR ligand-binding module were used to create the TetR-MphR. This resulting hybrid regulator possesses DNA-binding properties of TetR and ligand response properties of MphR, which is able to control gene expression in response to a molecular signal in cells. Furthermore, we studied molecular interactions between the TetR DNA-binding module and MphR ligand-binding module by using mutant analysis. Together, we demonstrated that TetR family regulators contain discrete and functional modules that can be used to build biological components with novel properties. This work highlights the utility of rational design as a means of creating modular parts for cell engineering and introduces new possibilities in rewiring cellular response pathways.

Encapsulation of Active Enzymes within Bacteriophage P22 Virus-Like Particles.

Virus-like particles (VLPs) are nonpathogenic protein cage structures derived from viral coat proteins that have found utility in the area of biomaterials and nanotechnology. VLPs have been exploited as containers for the sequestration and encapsulation of a wide range of guest molecules in their hollow interiors. The robust nature of VLPs lend them as versatile scaffolds that can be exploited to provide protection to encapsulated guest molecules, such as enzymes which are often susceptible to inactivation and degradation, and for organization and construction of new nanomaterials incorporating the chemical properties of the guest molecules. In this chapter a background and methodology for the encapsulation of enzymes on the interior of the bacteriophage P22 derived VLP is described.

Modular Self-Assembly of Protein Cage Lattices for Multistep Catalysis.

The assembly of individual molecules into hierarchical structures is a promising strategy for developing three-dimensional materials with properties arising from interaction between the individual building blocks. Virus capsids are elegant examples of biomolecular nanostructures, which are themselves hierarchically assembled from a limited number of protein subunits. Here, we demonstrate the bio-inspired modular construction of materials with two levels of hierarchy: the formation of catalytically active individual virus-like particles (VLPs) through directed self-assembly of capsid subunits with enzyme encapsulation, and the assembly of these VLP building blocks into three-dimensional arrays. The structure of the assembled arrays was successfully altered from an amorphous aggregate to an ordered structure, with a face-centered cubic lattice, by modifying the exterior surface of the VLP without changing its overall morphology, to modulate interparticle interactions. The assembly behavior and resultant lattice structure was a consequence of interparticle interaction between exterior surfaces of individual particles and thus independent of the enzyme cargos encapsulated within the VLPs. These superlattice materials, composed of two populations of enzyme-packaged VLP modules, retained the coupled catalytic activity in a two-step reaction for isobutanol synthesis. This study demonstrates a significant step toward the bottom-up fabrication of functional superlattice materials using a self-assembly process across multiple length scales and exhibits properties and function that arise from the interaction between individual building blocks.

Self-assembling biomolecular catalysts for hydrogen production.

The chemistry of highly evolved protein-based compartments has inspired the design of new catalytically active materials that self-assemble from biological components. A frontier of this biodesign is the potential to contribute new catalytic systems for the production of sustainable fuels, such as hydrogen. Here, we show the encapsulation and protection of an active hydrogen-producing and oxygen-tolerant [NiFe]-hydrogenase, sequestered within the capsid of the bacteriophage P22 through directed self-assembly. We co-opted Escherichia coli for biomolecular synthesis and assembly of this nanomaterial by expressing and maturing the EcHyd-1 hydrogenase prior to expression of the P22 coat protein, which subsequently self assembles. By probing the infrared spectroscopic signatures and catalytic activity of the engineered material, we demonstrate that the capsid provides stability and protection to the hydrogenase cargo. These results illustrate how combining biological function with directed supramolecular self-assembly can be used to create new materials for sustainable catalysis.

Viruslike Particles Encapsidating Respiratory Syncytial Virus M and M2 Proteins Induce Robust T Cell Responses.

Subunit vaccines provide a safe, focused alternative to conventional vaccines. However, these vaccines often require significant adjuvants and are particularly hard to target toward cytotoxic T lymphocyte (CTL) immunity. Viruslike particles (VLPs) provide biomaterial scaffolds with pathogen-like polyvalent structures making them useful platforms for biomimetic antigen delivery to the immune system. Encapsidation of antigens within VLPs has been shown to enhance antigen availability for CD8 T cell responses. Here, we examine the potential to generate complex responses to multiple subunit antigens localized within the same VLP particle. Two proteins of respiratory syncytial virus (RSV) with well-characterized CD8 T cell responses, the matrix (M) and matrix 2 (M2) proteins, were successfully coencapsidated within the P22 VLP. Upon intranasal administration in mice, the particles stimulated CD8 T cell memory responses against both antigens. In addition, vaccination elicited tissue-resident T cell populations. Upon subsequent RSV challenge, P22-M/M2-treated mice displayed significantly reduced lung viral titers. This demonstrates the utility of the P22 VLP in directing immune responses to multiple encapsidated viral antigens, demonstrating the potential of this technology to facilitate immunity to multiple targets simultaneously.

Design of a VLP-nanovehicle for CYP450 enzymatic activity delivery.

BACKGROUND: The intracellular delivery of enzymes for therapeutic use has a promising future for the treatment of several diseases such as genetic disorders and cancer. Virus-like particles offer an interesting platform for enzymatic delivery to targeted cells because of their great cargo capacity and the enhancement of the biocatalyst stability towards several factors important in the practical application of these nanoparticles. RESULTS: We have designed a nano-bioreactor based on the encapsulation of a cytochrome P450 (CYP) inside the capsid derived from the bacteriophage P22. An enhanced peroxigenase, CYPBM3, was selected as a model enzyme because of its potential in enzyme prodrug therapy. A total of 109 enzymes per capsid were encapsulated with a 70 % retention of activity for cytochromes with the correct incorporation of the heme cofactor. Upon encapsulation, the stability of the enzyme towards protease degradation and acidic pH was increased. Cytochrome P450 activity was delivered into Human cervix carcinoma cells via transfecting P22-CYP nanoparticles with lipofectamine. CONCLUSION: This work provides a clear demonstration of the potential of biocatalytic virus-like particles as medical relevant enzymatic delivery vehicles for clinical applications.

Virus-like particles as antigenic nanomaterials for inducing protective immune responses in the lung.

The lung is a major entry point for many of the most detrimental pathogens to human health. The onslaught of pathogens encountered by the lung is counteracted by protective immune responses that are generated locally, which can be stimulated through vaccine strategies to prevent pathogen infections. Here, we discuss the use of virus-like particles (VLPs), nonpathogen derivatives of viruses or protein cage structures, to construct new vaccines exploiting the lung as a site for immunostimulation. VLPs are unique in their ability to be engineered with near molecular level detail and knowledge of their composition and structure. A summary of research in developing VLP-based vaccines for the lung is presented that suggests promising results for future vaccine development.

Characterization of a highly flexible self-assembling protein system designed to form nanocages.

The design of proteins that self-assemble into well-defined, higher order structures is an important goal that has potential applications in synthetic biology, materials science, and medicine. We previously designed a two-component protein system, designated A-(+) and A-(-), in which self-assembly is mediated by complementary electrostatic interactions between two coiled-coil sequences appended to the C-terminus of a homotrimeric enzyme with C3 symmetry. The coiled-coil sequences are attached through a short, flexible spacer sequence providing the system with a high degree of conformational flexibility. Thus, the primary constraint guiding which structures the system may assemble into is the symmetry of the protein building block. We have now characterized the properties of the self-assembling system as a whole using native gel electrophoresis and analytical ultracentrifugation (AUC) and the properties of individual assemblies using cryo-electron microscopy (EM). We show that upon mixing, A-(+) and A-(-) form only six different complexes in significant concentrations. The three predominant complexes have hydrodynamic properties consistent with the formation of heterodimeric, tetrahedral, and octahedral protein cages. Cryo-EM of size-fractionated material shows that A-(+) and A-(-) form spherical particles with diameters appropriate for tetrahedral or octahedral protein cages. The particles varied in diameter in an almost continuous manner suggesting that their structures are extremely flexible.

Encapsulation of an enzyme cascade within the bacteriophage P22 virus-like particle.

Developing methods for investigating coupled enzyme systems under conditions that mimic the cellular environment remains a significant challenge. Here we describe a biomimetic approach for constructing densely packed and confined multienzyme systems through the co-encapsulation of 2 and 3 enzymes within a virus-like particle (VLP) that perform a coupled cascade of reactions, creating a synthetic metabolon. Enzymes are efficiently encapsulated in vivo with known stoichiometries, and the kinetic parameters of the individual and coupled activities are characterized. From the results we develop and validate a mathematical model for predicting the expected kinetics for coupled reactions under co-localized conditions.

Constructing catalytic antimicrobial nanoparticles by encapsulation of hydrogen peroxide producing enzyme inside the P22 VLP.

Here we examine a self-assembling virus like particle to construct catalytically active nanoparticles that can inhibit bacterial growth. The results suggest that encapsulation of enzymes inside VLPs can be exploited to develop new bionanomaterials with useful functionalities.

Rescuing recombinant proteins by sequestration into the P22 VLP.

Here we report the use of a self-assembling protein cage to sequester and solubilize recombinant proteins which are usually trafficked to insoluble inclusion bodies. Our results suggest that protein cages can be used as novel vehicles to rescue and produce soluble proteins that are otherwise difficult to obtain using conventional methods.

Biomimetic antigenic nanoparticles elicit controlled protective immune response to influenza.

Here we present a biomimetic strategy toward nanoparticle design for controlled immune response through encapsulation of conserved internal influenza proteins on the interior of virus-like particles (VLPs) to direct CD8+ cytotoxic T cell protection. Programmed encapsulation and sequestration of the conserved nucleoprotein (NP) from influenza on the interior of a VLP, derived from the bacteriophage P22, results in a vaccine that provides multistrain protection against 100 times lethal doses of influenza in an NP specific CD8+ T cell-dependent manner. VLP assembly and encapsulation of the immunogenic NP cargo protein is the result of a genetically programmed self-assembly making this strategy amendable to the quick production of vaccines to rapidly emerging pathogens. Addition of adjuvants or targeting molecules were not required for eliciting the protective response.

Nanoreactors by programmed enzyme encapsulation inside the capsid of the bacteriophage P22.

The virus like particle (VLP) derived from bacteriophage P22 presents a unique platform for constructing catalytically functional nanomaterials by encapsulation of enzymes into its interior. Encapsulation has been engineered to be genetically programmed allowing "one pot" synthesis and incorporation of desired enzymes. The unique characteristic that separates P22 from other VLP systems is the ability to modulate the overall volume and porosity of the VLP structure, thus controlling substrate access to the encapsulated enzyme. The present study demonstrates incorporation of an enzyme, alcohol dehydrogenase D, with the highest internal loading for an active enzyme by any VLP described thus far. In addition, we show that not only does encapsulating AdhD inside P22 affect its kinetic parameters in comparison with the "free" enzyme, but transformation of P22 to different morphological states, which changes the internal volume of the VLP, yields changes in the overall activity of the encapsulated enzyme as well. The findings reported here clearly illustrate that P22 holds potential for synthetic approaches to create nanoreactors, by design, using the power of highly evolved enzymes for chemical transformations.

Evaluation of a symmetry-based strategy for assembling protein complexes.

We evaluate a strategy for assembling proteins into large cage-like structures, based on the symmetry associated with the native protein's quaternary structure. Using a trimeric protein, KDPG aldolase, as a building block, two fusion proteins were designed that could assemble together upon mixing. The fusion proteins, designated A-(+) and A-(-), comprise the aldolase domain, a short, flexible spacer sequence, and a sequence designed to form a heterodimeric antiparallel coiled-coil between A-(+) and A-(-). The flexible spacer is included to minimize constraints on the ability of the fusion proteins to assemble into larger structures. On incubating together, A-(+) and A-(-) assembled into a mixture of complexes that were analyzed by size exclusion chromatography coupled to multi-angle laser light scattering, analytical ultracentrifugation, transmission electron microscopy and atomic force microscopy. Our analysis indicates that, despite the inherent flexibility of the assembly strategy, the proteins assemble into a limited number of globular structures. Dimeric and tetrameric complexes of A-(+) and A-(-) predominate, with some evidence for the formation of larger assemblies; e.g. octameric A-(+): A-(-) complexes.

Adenosyl radical: reagent and catalyst in enzyme reactions.

Adenosine is undoubtedly an ancient biological molecule that is a component of many enzyme cofactors: ATP, FADH, NAD(P)H, and coenzyme A, to name but a few, and, of course, of RNA. Here we present an overview of the role of adenosine in its most reactive form: as an organic radical formed either by homolytic cleavage of adenosylcobalamin (coenzyme B(12), AdoCbl) or by single-electron reduction of S-adenosylmethionine (AdoMet) complexed to an iron-sulfur cluster. Although many of the enzymes we discuss are newly discovered, adenosine's role as a radical cofactor most likely arose very early in evolution, before the advent of photosynthesis and the production of molecular oxygen, which rapidly inactivates many radical enzymes. AdoCbl-dependent enzymes appear to be confined to a rather narrow repertoire of rearrangement reactions involving 1,2-hydrogen atom migrations; nevertheless, mechanistic insights gained from studying these enzymes have proved extremely valuable in understanding how enzymes generate and control highly reactive free radical intermediates. In contrast, there has been a recent explosion in the number of radical-AdoMet enzymes discovered that catalyze a remarkably wide range of chemically challenging reactions; here there is much still to learn about their mechanisms. Although all the radical-AdoMet enzymes so far characterized come from anaerobically growing microbes and are very oxygen sensitive, there is tantalizing evidence that some of these enzymes might be active in aerobic organisms including humans.

Subunit structure of benzylsuccinate synthase.

Benzylsuccinate synthase is a member of the glycyl radical family of enzymes. It catalyzes the addition of toluene to fumarate to form benzylsuccinate as the first step in the anaerobic pathway of toluene fermentation. The enzyme comprises three subunits, alpha, beta, and gamma, that in Thauera aromatica strain T1 are encoded by the tutD, tutG, and tutF genes, respectively. The large alpha-subunit contains the essential glycine and cysteine residues that are conserved in all glycyl radical enzymes. However, the function of the small beta- and gamma-subunits has remained unclear. We have overexpressed all three subunits of benzylsuccinate synthase in Escherichia coli, both individually and in combination. Coexpression of the gamma-subunit (but not the beta-subunit) is essential for efficient expression of the alpha-subunit. The benzylsuccinate synthase complex lacking the glycyl radical could be purified as an alpha(2)beta(2)gamma(2) hexamer by nickel affinity chromatography through a "His(6)" affinity tag engineered onto the C-terminus of the alpha-subunit. Unexpectedly, BSS was found to contain two iron-sulfur clusters, one associated with the beta-subunit and the other with the gamma-subunit that appear to be necessary for the structural integrity of the complex. The spectroscopic properties of these clusters suggest that they are most likely [4Fe-4S] clusters. Removal of iron with chelating agents results in dissociation of the complex; similarly, a mutant gamma-subunit lacking the [4Fe-4S] cluster is unable to stabilize the alpha-subunit when the proteins are coexpressed.