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.

Encapsulation of Pseudomonas aeruginosa elastase inside the P22 virus-like particle for controlling enzyme-substrate interactions.

Controlling interactions between enzymes and interaction partners, such as substrates, is important for applications in cellular biology and molecular biochemistry. A strategy for controlling enzyme access with substrate interaction partners is to exploit encapsulation of enzymes inside nanoparticles to limit the accessibility of the enzymes to large macromolecules, but allow free exchange of small-molecule substrates. The research here evaluates the encapsulation of Pseudomonas aeruginosa elastase inside the bacteriophage P22 virus-like particle (VLP) to examine the ability to allow free soluble substrates access to the enzyme while blocking large macromolecular substrate interactions.

Engineering soluble artificial epidermal growth factor receptor mimics capable of spontaneous in vitro dimerization.

Epidermal growth factor receptor (EGFR) is a clinically validated target for a multitude of human cancers. The receptor is activated upon ligand binding through a critical dimerization step. Dimerization can be replicated in vitro by locally concentrating the receptor kinase domains on the surface of lipid-based vesicles.

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).

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.

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.

A Superior Synthesis of Longitudinally Twisted Acenes.

Seven longitudinally twisted acenes (an anthracene, two tetracenes, three pentacenes, and a hexacene) have been synthesized by the addition of aryllithium reagents to the appropriate quinone precursors, followed by SnCl -mediated reduction of their diol intermediates, and several of these acenes have been crystallographically characterized. The new syntheses of the three previously reported twisted acenes, decaphenylanthracene (1), 9,10,11,20,21,22-hexaphenyltetrabenzo[a,c,l,n]pentacene (2), and 9,10,11,12,13,14,15,16-octaphenyldibenzo[a,c]tetracene (14), resulted in a reduction of the number of synthetic steps. As a consequence their overall yields were increased by factors of 50-, 24-, and 66-fold, respectively.

Sortase-Mediated Ligation as a Modular Approach for the Covalent Attachment of Proteins to the Exterior of the Bacteriophage P22 Virus-like Particle.

Virus-like particles are unique platforms well suited for the construction of nanomaterials with broad-range applications. The research presented here describes the development of a modular approach for the covalent attachment of protein domains to the exterior of the versatile bacteriophage P22 virus-like particle (VLP) via a sortase-mediated ligation strategy. The bacteriophage P22 coat protein was genetically engineered to incorporate an LPETG amino acid sequence on the C-terminus, providing the peptide recognition sequence utilized by the sortase enzyme to catalyze peptide bond formation between the LPETG-tagged protein and a protein containing a polyglycine sequence on the N-terminus.

Two-Dimensional Crystallization of P22 Virus-Like Particles.

Virus-like particles (VLPs) are well established platforms for constructing functional biomimetic materials. The VLP from the bacteriophage P22 can be used as a nanocontainer to sequester active enzymes, at high concentration, within its cavity through a process of directed self-assembly. Construction of ordered 2D assemblies of these catalytic VLPs can be envisioned as a functional membrane.

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.

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.

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.

Large area synthesis of a nanoporous two-dimensional polymer at the air/water interface.

We present the synthesis of a two-dimensional polymer at the air/water interface and its nm-resolution imaging. Trigonal star, amphiphilic monomers bearing three anthraceno groups on a central triptycene core are confined at the air/water interface. Compression followed by photopolymerization on the interface provides the two-dimensional polymer.

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.

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.

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.

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.

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.

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.

Intramolecular hydrogen bonding as a synthetic tool to induce chemical selectivity in acid catalyzed porphyrin synthesis.

A straightforward procedure based on the formation of intramolecular hydrogen bonds to impart selectivity in the preparation of multi-functionalized porphyrins has been developed. To illustrate the concept, the synthesis of a biomimetic artificial photosynthetic model able to undergo electron and proton transfer reactions upon irradiation is reported.

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-(-).

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.

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.