Chunxi Hou

Construction of GPx Active Centers on Natural Protein Nanodisk/Nanotube: A New Way to Develop Artificial Nanoenzyme

Construction of catalytic centers on natural protein aggregates is a challenging topic in biomaterial and biomedicine research. Here we report a novel construction of artificial nanoenzyme with glutathione peroxidase (GPx)-like function. By engineering the surface of tobacco mosaic virus (TMV) coat protein, the main catalytic components of GPx were fabricated on TMV protein monomers. Through direct self-assembly of the functionalized viral coat proteins, the multi-GPx centers were installed on these well-defined nanodisks or nanotubes. With the help of muti-selenoenzyme centers, the resulting organized nanoenzyme exhibited remarkable GPx activity, even approaching the level of natural GPx. The antioxidation study on subcell mitochondrial level demonstrated that virus-based nanoenzyme exerted excellent capacity for protecting cell from oxidative damage. This strategy represents a new way to develop artificial nanoenzymes.

Quantum-Dot-Induced Self-Assembly of Cricoid Protein for Light Harvesting

Stable protein one (SP1) has been demonstrated as an appealing building block to design highly ordered architectures, despite the hybrid assembly with other nano-objects still being a challenge. Herein, we developed a strategy to construct high-ordered protein nanostructures by electrostatic self-assembly of cricoid protein nanorings and globular quantum dots (QDs). Using multielectrostatic interactions between 12mer protein nanoring SP1 and oppositely charged CdTe QDs, highly ordered nanowires with sandwich structure were achieved by hybridized self-assembly. QDs with different sizes (QD1, 3-4 nm; QD2, 5-6 nm; QD3, ∼10 nm) would induce the self-assembly protein rings into various nanowires, subsequent bundles, and irregular networks in aqueous solution. Atomic force microscopy, transmission electron microscopy, and dynamic light scattering characterizations confirmed that the size of QDs and the structural topology of the nanoring play critical functions in the formation of the superstructures. Furthermore, an ordered arrangement of QDs provides an ideal scaffold for designing the light-harvesting antenna. Most importantly, when different sized QDs (e.g., QD1 and QD3) self-assembled with SP1, an extremely efficient Förster resonance energy transfer was observed on these protein nanowires. The self-assembled protein nanostructures were demonstrated as a promising scaffold for the development of an artificial light-harvesting system.

A Dual Enzyme Microgel with High Antioxidant Ability Based on Engineered Seleno‐Ferritin and Artificial Superoxide Dismutase

An antioxidant microgel with both glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities is reported. Using computational design and genetic engineering methods, the main catalytic components of GPx are fabricated onto the surface of ferritin. The resulting seleno-ferritin (Se-Fn) monomers can self-assemble into nanocomposites that exhibit remarkable GPx activity due to the well organized multi-GPx catalytic centers. Subsequently, a porphyrin derivative is synthesized as an SOD mimic, and is employed to construct a synergistic dual enzyme system by crosslinking Se-Fn nanocomposites into a microgel. Significantly, this dual enzyme microgel is demonstrated to display better antioxidant ability than single GPx or SOD mimics in protecting cells from oxidative damage.

Enzyme-Triggered Defined Protein Nanoarrays: Efficient Light-Harvesting Systems to Mimic Chloroplasts

The elegance and efficiency by which chloroplasts harvest solar energy and conduct energy transfer have been a source of inspiration for chemists to mimic such process. However, precise manipulation to obtain orderly arranged antenna chromophores in constructing artificial chloroplast mimics was a great challenge, especially from the structural similarity and bioaffinity standpoints. Here we reported a design strategy that combined covalent and noncovalent interactions to prepare a protein-based light-harvesting system to mimic chloroplasts. Cricoid stable protein one (SP1) was utilized as a building block model. Under enzyme-triggered covalent protein assembly, mutant SP1 with tyrosine (Tyr) residues at the designated sites can couple together to form nanostructures. Through controlling the Tyr sites on the protein surface, we can manipulate the assembly orientation to respectively generate 1D nanotubes and 2D nanosheets. The excellent stability endowed the self-assembled protein architectures with promising applications. We further integrated quantum dots (QDs) possessing optical and electronic properties with the 2D nanosheets to fabricate chloroplast mimics. By attaching different sized QDs as donor and acceptor chromophores to the negatively charged surface of SP1-based protein nanosheets via electrostatic interactions, we successfully developed an artificial light-harvesting system. The assembled protein nanosheets structurally resembled the natural thylakoids, and the QDs can achieve pronounced FRET phenomenon just like the chlorophylls. Therefore, the coassembled system was meaningful to explore the photosynthetic process in vitro, as it was designed to mimic the natural chloroplast.

Triple mutated antibody scFv2F3 with high GPx activity: insights from MD, docking, MDFE, and MM-PBSA simulation

By combining computational design and site-directed mutagenesis, we have engineered a new catalytic ability into the antibody scFv2F3 by installing a catalytic triad (Trp29–Sec52–Gln72). The resulting abzyme, Se-scFv2F3, exhibits a high glutathione peroxidase (GPx) activity, approaching the native enzyme activity. Activity assays and a systematic computational study were performed to investigate the effect of successive replacement of residues at positions 29, 52, and 72. The results revealed that an active site Ser52/Sec substitution is critical for the GPx activity of Se-scFv2F3. In addition, Phe29/Trp–Val72/Gln mutations enhance the reaction rate via functional cooperation with Sec52. Molecular dynamics simulations showed that the designed catalytic triad is very stable and the conformational flexibility caused by Tyr101 occurs mainly in the loop of complementarity determining region 3. The docking studies illustrated the importance of this loop that favors the conformational shift of Tyr54, Asn55, and Gly56 to stabilize substrate binding. Molecular dynamics free energy and molecular mechanics-Poisson Boltzmann surface area calculations estimated the pKa shifts of the catalytic residue and the binding free energies of docked complexes, suggesting that dipole–dipole interactions among Trp29–Sec52–Gln72 lead to the change of free energy that promotes the residual catalytic activity and the substrate-binding capacity. The calculated results agree well with the experimental data, which should help to clarify why Se-scFv2F3 exhibits high catalytic efficiency.

Supramolecular Protein Assemblies Based on DNA Templates

DNA plays an important role in the process of protein assembly. DNA viruses such as the M13 virus are typical examples in which single DNA genomes behave as templates to induce the assembly of multiple major coat protein (PVIII) monomers. Thus, the design of protein assemblies based on DNA templates attracts much interest in the construction of supramolecular structures and materials. With the development of DNA nanotechnology, precise 1D and 3D protein nanostructures have been designed and constructed by using DNA templates through DNA-protein interactions, protein-ligand interactions, and protein-adapter interactions. These DNA-templated protein assemblies show great potential in catalysis, medicine, light-responsive systems, drug delivery, and signal transduction. Herein, we review the progress on DNA-based protein nanostructures that possess sophisticated nanometer-sized structures with programmable shapes and stimuli-responsive parameters, and we present their great potential in the design of biomaterials and biodevices in the future.

Photocontrolled protein assembly for constructing programmed two-dimensional nanomaterials

Precise self-assembly of proteins with structural heterogeneity, flexibility, and complexity into programmed arrays to mimic the exquisite architectures created by Nature is a great challenge for the development of protein-based functional nanomaterials. Herein, we present a strategy that integrates light stimuli and covalent coupling to prepare size-tunable two-dimensional (2D) protein nanostructures by remote photocontrol. Using Ru(bpy)3 2+ as a photosensitizer, stable protein one (SP1) was redesigned and self-assembled into nanosheets in the presence of ammonium persulfate (APS) through a rapid and efficient oxidative protein crosslinking reaction. In the design, only a serine-to-tyrosine mutation at position 98 was introduced into SP1 by combining computer simulation and genetic engineering for specific covalent coupling under white light illumination. The chemical and topographical specificities of the photosensitized crosslinking reaction allow control of the direction of protein assembly to form extended 2D nanosheets, which are packed in an orderly manner along the lateral surface of ring-shaped SP1S98Y. Notably, the growth of SP1 nanosheets exhibited isotropical characteristics and can be dynamically mediated by illumination time to achieve precise control of the size of the assembled architectures. The subsequent heat treatment further revealed the excellent thermostability of the 2D periodic SP1 nanostructures, which may find promising applications in the fabrication of various nanobiomaterials after functionalization. The present work demonstrates that the visible light-triggered crosslinking strategy is a facile and environmentally friendly method for constructing advanced protein architectures through hierarchical self-assembly.

Reductive-Responsive Single Molecular Layer Polymer Nanocapsules Prepared by Lateral Functionalized Pillar[5]arenes for Targeting Anticancer Drug Delivery

Herein, a new reductive-responsive pillar[5]arene-based, single-molecule-layer polymer nanocapsule is constructed for drug delivery. The functionalized system shows good biocompatibility, efficient internalization into targeted cells and obvious triggered release of entrapped drugs in a reducing environment such as cytoplasm. Besides, this smart vehicle loaded with anticancer drug shows excellent inhibition for tumor cell proliferation and exhibits low side effect on normal cells. This work not only demonstrates the development of a new reductive-responsive single molecular layer polymer nanocapsule for anticancer drug targeting delivery but also extends the design of smart materials for biomedical applications.