Fusion then fission: splitting and reassembly of an artificial fusion-protein nanocage.

A split-protein system is a simple approach to introduce new termini which are useful as modification sites in protein engineering, but has been adapted mainly for monomeric proteins. Here we demonstrate the design of split subunits of the 60-mer artificial fusion-protein nanocage TIP60. The subunit fragments successfully reformed the cage structure in the same manner as prior to splitting.

Thermally Reversible Gel-Sol Transition of Hydrogels via Dissociation and Association of an Artificial Protein Nanocage.

Oligomeric protein nanocages often disassemble into their subunits and reassemble by external stimuli. Thus, using these nanocages as cross-linkers for hydrogel network structures is a promising approach to allow hydrogels to undergo stimuli-responsive gel-sol transitions or self-healing. Here, we report hydrogels that show a reversible gel-sol transition resulting from the heat-induced dissociation and reassociation of protein nanocages.

Dual Modification of Artificial Protein Cage.

Chemical modifications of proteins confer new functions on them or modulate their original functions. Although various approaches are developed for modifications, modifications of the two different reactive sites of proteins by different chemicals are still challenging. In this chapter, we show a simple approach for selective modifications of both interior and exterior surfaces of protein nanocages by two different chemicals based on a molecular size filter effect of the surface pores.

Hydrophobization of a TIP60 Protein Nanocage for the Encapsulation of Hydrophobic Compounds.

Encapsulation of hydrophobic molecules in protein-based nanocages is a promising approach for dispersing these molecules in water. Here, we report a chemical modification approach to produce a protein nanocage with a hydrophobic interior surface based on our previously developed nanocage, TIP60. The large pores of TIP60 act as tunnels for small molecules, allowing modification of the interior surface by hydrophobic compounds without nanocage disassembly.

Column-free purification of an artificial protein nanocage, TIP60.

Protein nanocages, which have inner cavities and surface pores, are attractive materials for various applications, such as in catalysts and medicine. Recently, we produced an artificial protein nanocage, TIP60, and demonstrated its potential as a stimuli-responsive nanocarrier. In the present study, we report a simple purification method for TIP60 that can replace time-consuming and costly affinity chromatography purification.

Reversible Assembly of an Artificial Protein Nanocage Using Alkaline Earth Metal Ions.

Protein nanocages are of increasing interest for use as drug capsules, but the encapsulation and release of drug molecules at appropriate times require the reversible association and dissociation of the nanocages. One promising approach to addressing this challenge is the design of metal-dependent associating proteins. Such designed proteins typically have Cys or His residues at the protein surface for connecting the associating proteins through metal-ion coordination.

Efficient Degradation of Poly(ethylene terephthalate) with Thermobifida fusca Cutinase Exhibiting Improved Catalytic Activity Generated using Mutagenesis and Additive-based Approaches.

Cutinases are promising agents for poly(ethylene terephthalate) (PET) bio-recycling because of their ability to produce the PET monomer terephthalic acid with high efficiency under mild reaction conditions. In this study, we found that the low-crystallinity PET (lcPET) hydrolysis activity of thermostable cutinase from Thermobifida fusca (TfCut2), was increased by the addition of cationic surfactant that attracts enzymes near the lcPET film surface via electrostatic interactions. This approach was applicable to the mutant TfCut2 G62A/F209A, which was designed based on a sequence comparison with PETase from Ideonella sakaiensis.

Acceleration of Enzymatic Degradation of Poly(ethylene terephthalate) by Surface Coating with Anionic Surfactants.

Enzymatic degradation of poly(ethylene terephthalate) (PET) is promising because this process is safer than conventional industrial approaches. Recently, a cationic PET hydrolase (PETase) was identified from Ideonella sakaiensis. Pre-incubation of a low-crystallinity PET film with anionic surfactants prior to initiating the reaction was found to improve PETase activity 120-fold.

Design of Hollow Protein Nanoparticles with Modifiable Interior and Exterior Surfaces.

Protein-based nanoparticles hold promise for a broad range of applications. Here, we report the production of a uniform anionic hollow protein nanoparticle, designated TIP60, which spontaneously assembles from a designed fusion protein subunit based on the geometric features of polyhedra. We show that TIP60 tolerates mutation and both its interior and exterior surfaces can be chemically modified.

Protein Nanoparticle Formation Using a Circularly Permuted α-Helix-Rich Trimeric Protein.

We here report the production of highly spherical protein nanoparticles based on the domain-swapping oligomerization of a circularly permuted trimeric protein, major histocompatibility complex (MHC) class II associated chaperonin. The size distribution of the nanoparticles can be adjusted to between 40 and 100 nm in diameter, and thus, these particles are suitable as drug carriers following purification under basic conditions. Our approach involves no harsh treatments and could provide an alternative approach for protein nanoparticle formation.

Draft Genome Sequence of Bordetella bronchiseptica KU1201, the First Isolation Source of Arylmalonate Decarboxylase.

The analysis of the 6.8-Mbp draft genome sequence of the phenylmalonate-assimilating bacterium Bordetella bronchiseptica KU1201 identified 6,358 protein-coding sequences. This will give us an insight into the catabolic variability of this strain for aromatic compounds, along with the roles of arylmalonate decarboxylases in nature.

Single-step reconstitution of apo-hemoproteins at the disruption stage of Escherichia coli cells.

Hemoproteins on their metal: We report a novel strategy for the reconstitution of hemoproteins with non-natural metal complexes; simple addition of manganese and ruthenium porphyrin to E. coli cells immediately prior to homogenization yields the reconstituted proteins. We believe that this simple approach could become a standard reconstitution method for hemoproteins.

Chiral-substrate-assisted stereoselective epoxidation catalyzed by H2O2-dependent cytochrome P450SPα.

The stereoselective epoxidation of styrene was catalyzed by H(2) O(2) -dependent cytochrome P450(SPα) in the presence of carboxylic acids as decoy molecules. The stereoselectivity of styrene oxide could be altered by the nature of the decoy molecules. In particular, the chirality at the α-positions of the decoy molecules induced a clear difference in the chirality of the product: (R)-ibuprofen enhanced the formation of (S)-styrene oxide, whereas (S)-ibuprofen preferentially afforded (R)-styrene oxide.

Characterization of vanadium-binding sites of the vanadium-binding protein Vanabin2 by site-directed mutagenesis.

Background: Vanabins are a unique protein family of vanadium-binding proteins with nine disulfide bonds. Possible binding sites for VO2+ in Vanabin2 from a vanadium-rich ascidian Ascidia sydneiensis samea have been detected by nuclear magnetic resonance study, but the metal selectivity and metal-binding ability of each site was not examined.

Methods: In order to reveal functional contribution of each binding site, we prepared several mutants of Vanabin2 by in vitro site-directed mutagenesis and analyzed their metal selectivity and affinity by immobilized metal-ion affinity chromatography and Hummel Dreyer method.

A novel vanadium reductase, Vanabin2, forms a possible cascade involved in electron transfer.

The unusual ascidian ability to accumulate high levels of vanadium ions at concentrations of up to 350 mM, a 10(7)-fold increase over that found in seawater, has been attracting interdisciplinary attention for a century. Accumulated V(V) is finally reduced to V(III) via V(IV) in ascidian vanadocytes. Reducing agents must therefore participate in the reduction.

Selective metal binding by Vanabin2 from the vanadium-rich ascidian, Ascidia sydneiensis samea.

Vanadium-binding proteins, or Vanabins, have recently been isolated from the vanadium-rich ascidian, Ascidia sydneiensis samea. Recent reports indicate that Vanabin2 binds twenty V(IV) ions at pH 7.5, and that it has a novel bow-shaped conformation.