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. One of the newly introduced terminals at the interior surface can be modified using a tag peptide and green fluorescent protein. Therefore, the termini could serve as a versatile modification site for incorporating a wide variety of functional peptides and proteins.

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. The hydrogel contained the 60-mer artificial protein nanocage, TIP60, as a supramolecular cross-linker for polyethylene glycol network structures. The hydrogel showed a gel-to-sol transition upon heating at a temperature above the melting point of TIP60 and immediately returned to a gel state upon cooling to room temperature. During the heating and cooling treatment of the hydrogel, small-angle X-ray scattering analysis suggested the dissociation and reassociation of TIP60. Furthermore, we demonstrated redox-responsive cargo release from TIP60 in the hydrogel. These results showed the potential of TIP60 as a component of multi-stimuli-responsive hydrogels.

Impedance-guided modified CLOSE protocol ablation can reduce ablation index necessary for pulmonary vein isolation in patients with atrial fibrillation.

BACKGROUND: Real-time monitoring of generator impedance drop is not considered in CLOSE protocol pulmonary vein (PV) isolation (PVI) in patients with atrial fibrillation (AF). We verified whether additional information of impedance drop could minimize ablation index required for PVI using modified CLOSE protocol (target ablation index ≥ 500 on anterior wall and ≥400 on posterior wall along with inter-lesion distance of 3-6 mm and maximum power of 35 W) without any adverse effect of procedural data and efficacy. METHODS: Sixty consecutive Japanese AF patients [paroxysmal AF: 43 (72 %) patients] underwent first-time PVI with modified CLOSE protocol with real-time monitoring of impedance drop (impedance-guided modified CLOSE protocol). Ablation tags were colored according to impedance drop and ablation was immediately terminated before reaching target ablation index if impedance drop of ≥10â€¯Ω was confirmed. Ablation index needed for PVI, first-pass PVI rate, other procedural data, and atrial tachyarrhythmia recurrence were evaluated. RESULTS: Mean ablation index and impedance drop on anterior and posterior walls were 437.6 ±â€¯43.5â€¯Ω and 10.2 ±â€¯2.6â€¯Ω and 393.3 ±â€¯27.4â€¯Ω and 9.3 ±â€¯2.2 Ω, respectively. First-pass PVI per PV pair was accomplished in 90/120 (75 %). No complications occurred. PV gaps after first-pass ablation were locationally most often found on right posterior wall than on the other parts (p < 0.001). There were no differences in mean contact force, impedance drop, and ablation index between walls with and without PV gaps after first-pass PV ablation. During a mean follow-up of 24 ±â€¯9 months, survival from atrial tachyarrhythmia recurrence was 51/60 (85 %) patients. CONCLUSIONS: Using additional generator impedance drop information may be useful to minimize radiofrequency current application to accomplish PVI with modified CLOSE protocol while maintaining efficacy and safety in Japanese AF population.

Multi-enzyme Machinery for Chitin Degradation in the Chitinolytic Bacterium Chitiniphilus shinanonensis SAY3T.

The chitinolytic bacterium, Chitiniphilus shinanonensis SAY3T was examined to characterize its chitin-degrading enzymes in view of its potential to convert biomass chitin into useful saccharides. A survey of the whole-genome sequence revealed 49 putative genes encoding polypeptides that are thought to be related to chitin degradation. Based on an analysis of the relative quantity of each transcript and an assay for chitin-degrading activity of recombinant proteins, a chitin degradation system driven by 19 chitinolytic enzymes was proposed. These include sixteen endo-type chitinases, two N-acetylglucosaminidases, and one lipopolysaccharide monooxygenase that catalyzes the oxidative cleavage of glycosidic bonds. Among the 16 chitinases, ChiL was characterized by its remarkable transglycosylation activity. Of the two N-acetylglucosaminidases (ChiI and ChiT), ChiI was the major enzyme, corresponding to > 98% of the total cellular activity. Surprisingly, a chiI-disrupted mutant was still able to grow on medium with powdered chitin or GlcNAc dimer. However, its growth rate was slightly lower compared to that of the wild-type SAY3. This multi-enzyme machinery composed of various types of chitinolytic enzymes may support SAY3 to efficiently utilize native chitin as a carbon and energy source and may play a role in developing an enzymatic process to decompose and utilize abundant chitin at the industrial scale.

Crystal structure and activity of a de novo enzyme, ferric enterobactin esterase Syn-F4.

Producing novel enzymes that are catalytically active in vitro and biologically functional in vivo is a key goal of synthetic biology. Previously, we reported Syn-F4, the first de novo protein that meets both criteria. Syn-F4 hydrolyzed the siderophore ferric enterobactin, and expression of Syn-F4 allowed an inviable strain of Escherichia coli (Δfes) to grow in iron-limited medium. Here, we describe the crystal structure of Syn-F4. Syn-F4 forms a dimeric 4-helix bundle. Each monomer comprises two long α-helices, and the loops of the Syn-F4 dimer are on the same end of the bundle (syn topology). Interestingly, there is a penetrated hole in the central region of the Syn-F4 structure. Extensive mutagenesis experiments in a previous study showed that five residues (Glu26, His74, Arg77, Lys78, and Arg85) were essential for enzymatic activity in vivo. All these residues are located around the hole in the central region of the Syn-F4 structure, suggesting a putative active site with a catalytic dyad (Glu26-His74). The complete inactivity of purified proteins with mutations at the five residues supports the putative active site and reaction mechanism. Molecular dynamics and docking simulations of the ferric enterobactin siderophore binding to the Syn-F4 structure demonstrate the dynamic property of the putative active site. The structure and active site of Syn-F4 are completely different from native enterobactin esterase enzymes, thereby demonstrating that proteins designed de novo can provide life-sustaining catalytic activities using structures and mechanisms dramatically different from those that arose in nature.

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.

One-pot synthesis of fibrillar-shaped functional nanomaterial using microbial transglutaminase.

Recently, functional nanowire production using amyloids as a scaffold for protein immobilization has attracted attention. However, protein immobilization on amyloid fibrils often caused protein inactivation. In this study, we investigated protein immobilization using enzymatic peptide ligation to suppress protein inactivation during immobilization. We attempted to immobilize functional molecules such as green fluorescent protein (GFP) and Nanoluc to a transthyretin (TTR) amyloid using microbial transglutaminase (MTG), which links the glutamine side chain to the primary amine. Linkage between amyloid fibrils and functional molecules was achieved through the MTG substrate sequence, and the functional molecules-loaded nanowires were successfully fabricated. We also found that the synthetic process from amyloidization to functional molecules immobilization could be achieved in a single-step procedure.All rights reserved.

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. However, Cys and His residues favor interactions with soft and borderline metal ions, such as Au+ and Zn2+, classified by the hard and soft acids and bases concept, restricting the types of metal ions available to drive association. Here, we show the alkaline earth (AE) metal-dependent association of the recently designed artificial protein nanocage TIP60, which is composed of 60-mer fusion proteins. The introduction of a Glu (hard base) mutation to the fusion protein (K67E mutant) prevented the formation of the 60-mer but formed the expected cage structure in the presence of Ca, Sr, or Ba ions (hard acids). Cryogenic electron microscopy (cryo-EM) analysis indicated a Ba ion at the interface of the subunits. Furthermore, we demonstrated the encapsulation and release of single-stranded DNA molecules using this system. Our results provide insights into the design of AE metal-dependent association and dissociation mechanisms for proteins.

Production of pentaglycine-fused proteins using Escherichia coli expression system without in vitro peptidase treatment.

Conjugation of functional molecules to peptides is necessary for protein analysis and applications. Transpeptidase sortase A catalyzes the ligation reaction between the amino acid sequence LPXTG and polyglycine and allows for peptide sequence-specific molecular modifications. In this study, the preparation of pentaglycine-fused green fluorescent protein (G5-GFP) via methionine truncation mediated by Escherichia coli endogenous methionyl aminopeptidase was investigated. Some expression vectors of GFP presenting MetGly5 at the N-terminal were constructed, and N-terminal sequence analyses of the protein expressed in E. coli were performed. When the first codon of the GFP-encoding sequence was AUG, a mixture of GFP without pentaglycine and G5-GFP was obtained. In contrast, when the first codon AUG was replaced with a codon encoding alanine, G5-GFP was obtained uniformly. These results showed that the location of AUG in the expression vector had a significant influence on the preparation of polyglycine-fused proteins. The obtained findings are useful for the preparation of polyglycine-fused substrates using E. coli.

Self-Assembling Lectin Nano-Block Oligomers Enhance Binding Avidity to Glycans.

Lectins, carbohydrate-binding proteins, are attractive biomolecules for medical and biotechnological applications. Many lectins have multiple carbohydrate recognition domains (CRDs) and strongly bind to specific glycans through multivalent binding effect. In our previous study, protein nano-building blocks (PN-blocks) were developed to construct self-assembling supramolecular nanostructures by linking two oligomeric proteins. A PN-block, WA20-foldon, constructed by fusing a dimeric four-helix bundle de novo protein WA20 to a trimeric foldon domain of T4 phage fibritin, self-assembled into several types of polyhedral nanoarchitectures in multiples of 6-mer. Another PN-block, the extender PN-block (ePN-block), constructed by tandemly joining two copies of WA20, self-assembled into cyclized and extended chain-type nanostructures. This study developed novel functional protein nano-building blocks (lectin nano-blocks) by fusing WA20 to a dimeric lectin, Agrocybe cylindracea galectin (ACG). The lectin nano-blocks self-assembled into various oligomers in multiples of 2-mer (dimer, tetramer, hexamer, octamer, etc.). The mass fractions of each oligomer were changed by the length of the linkers between WA20 and ACG. The binding avidity of the lectin nano-block oligomers to glycans was significantly increased through multivalent effects compared with that of the original ACG dimer. Lectin nano-blocks with high avidity will be useful for various applications, such as specific cell labeling.

Icosahedral 60-meric porous structure of designed supramolecular protein nanoparticle TIP60.

Supramolecular protein nanoparticles and nanocages have potential in a broad range of applications. Recently, we developed a uniform supramolecular protein nanoparticle, TIP60, symmmetrically self-assembled from fusion proteins of a pentameric Sm-like protein and a dimeric MyoX-coil domain. Herein, we report the icosahedral 60-meric structure of TIP60 solved using single-particle cryo-electron microscopy. Interestingly, the structure revealed 20 regular-triangle-like pores on the surface. TIP60 and its mutants have many modifiable sites on their exterior and interior surfaces. The TIP60 architecture will be useful in the development of biomedical and biochemical nanoparticles/nanocages for future applications.

Rational thermostabilisation of four-helix bundle dimeric de novo proteins.

The stability of proteins is an important factor for industrial and medical applications. Improving protein stability is one of the main subjects in protein engineering. In a previous study, we improved the stability of a four-helix bundle dimeric de novo protein (WA20) by five mutations. The stabilised mutant (H26L/G28S/N34L/V71L/E78L, SUWA) showed an extremely high denaturation midpoint temperature (Tm). Although SUWA is a remarkably hyperstable protein, in protein design and engineering, it is an attractive challenge to rationally explore more stable mutants. In this study, we predicted stabilising mutations of WA20 by in silico saturation mutagenesis and molecular dynamics simulation, and experimentally confirmed three stabilising mutations of WA20 (N22A, N22E, and H86K). The stability of a double mutant (N22A/H86K, rationally optimised WA20, ROWA) was greatly improved compared with WA20 (ΔTm = 10.6 °C). The model structures suggested that N22A enhances the stability of the α-helices and N22E and H86K contribute to salt-bridge formation for protein stabilisation. These mutations were also added to SUWA and improved its Tm. Remarkably, the most stable mutant of SUWA (N22E/H86K, rationally optimised SUWA, ROSA) showed the highest Tm (129.0 °C). These new thermostable mutants will be useful as a component of protein nanobuilding blocks to construct supramolecular protein complexes.

Design of helical linkers for fusion proteins and protein-based nanostructures.

The construction of recombinant fusion/chimeric proteins has been widely used for expression of soluble proteins and protein purification in a variety of fields of protein engineering and biotechnology. Fusion proteins are constructed by the linking of two protein domains with a peptide linker. The selection of a linker sequence is important for the construction of stable and bioactive fusion proteins. Empirically designed linkers are generally classified into two categories according to their structural features: flexible linkers and rigid linkers. Rigid linkers with the α-helix-forming sequences A(EAAAK)nA (n=2-5) were first designed about two decades ago to control the distance between two protein domains and to reduce their interference. Thereafter, the helical linkers have been applied to the construction of many fusion proteins to improve expression and bioactivity. In addition, the design of fusion proteins that self-assemble into supramolecular complexes is useful for nanobiotechnology and synthetic biology. A protein that forms a self-assembling oligomer was fused by a rigid helical linker to another protein that forms another self-assembling oligomer, and the fusion protein symmetrically self-assembled into a designed protein nanoparticle or nanomaterial. Moreover, to construct chain-like polymeric nanostructures, extender protein nanobuilding blocks were designed by tandemly fusing two dimeric de novo proteins with helical or flexible linkers. The linker design of fusion proteins can affect conformation and dynamics of self-assembling nanostructures. The present review and methods focus on useful helical linkers to construct bioactive fusion proteins and protein-based nanostructures.

Rhodeus flaviventris, a new bitterling (Teleostei: Cyprinidae: Acheilognathinae) from China.

Rhodeus flaviventris, a new species, is described from the Le-An River, a tributary flowing into Poyang Lake in the Yangtze River basin, in Jiangxi Province, China. It is distinguished from all congeners by the combination of characters: branched dorsal-fin rays 9 (9-10); branched anal-fin rays 9 (9-10); longest simple rays of dorsal and anal fins thick and stiff; longitudinal scale series 33-34; transverse scale series 11 (10-12); pored scales 4-7; vertebrae 32-34; spindle-shaped eggs with major axis of 3.1-3.7 mm; color pattern of nuptial males (iris yellowish, belly yellow, central part of caudal fin orange, dorsal and anal fins with reddish-orange stripe).

Hyperstable De Novo Protein with a Dimeric Bisecting Topology.

Recently, we designed and assembled protein nanobuilding blocks (PN-Blocks) from an intermolecularly folded dimeric de novo protein called WA20. Using this dimeric 4-helix bundle, we constructed a series of self-assembling supramolecular nanostructures including polyhedra and chain-type complexes. Here we describe the stabilization of WA20 by designing mutations that stabilize the helices and hydrophobic core. The redesigned proteins denature with substantially higher midpoints, with the most stable variant, called Super WA20 (SUWA), displaying an extremely high midpoint (Tm = 122 °C), much higher than the Tm of WA20 (75 °C). The crystal structure of SUWA reveals an intermolecularly folded dimer with bisecting U topology, similar to the parental WA20 structure, with two long α-helices of a protomer intertwined with the helices of another protomer. Molecular dynamics simulations demonstrate that the redesigned hydrophobic core in the center of SUWA significantly suppresses the deformation of helices observed in the same region of WA20, suggesting this is a critical factor stabilizing the SUWA structure. This hyperstable de novo protein is expected to be useful as nanoscale pillars of PN-Block components in new types of self-assembling nanoarchitectures.

Development of behavioural automaticity by extended Pavlovian training in an insect.

The effect of repetitive training on learned actions has been a major subject in behavioural neuroscience. Many studies of instrumental conditioning in mammals, including humans, suggested that learned actions early in training are goal-driven and controlled by outcome expectancy, but they become more automatic and insensitive to reduction in the value of the outcome after extended training. It was unknown, however, whether the development of value-insensitive behaviour also occurs by extended training of Pavlovian conditioning in any animals. Here we show that crickets Gryllus bimaculatus that had received minimal training to associate an odour with water (unconditioned stimulus, US) did not exhibit conditioned response (CR) to the odour when they were given water until satiation before the test, but those that had received extended training exhibited CR even when they were satiated with water. Further pharmacological experiments suggested that octopamine neurons, the invertebrate counterparts of noradrenaline neurons, mediate US value signals and control execution of CR after minimal training, but the control diminishes with the progress of training and hence the CR becomes insensitive to US devaluation. The results suggest that repetitive sensory experiences can lead to a change from a goal-driven response to a more automatic one in crickets.

Lectin engineering: the possible and the actual.

Lectins are a widespread group of sugar-binding proteins occurring in all types of organisms including animals, plants, bacteria, fungi and even viruses. According to a recent report, there are more than 50 lectin scaffolds (∼Pfam), for which three-dimensional structures are known and sugar-binding functions have been confirmed in the literature, which far exceeds our view in the twentieth century (Fujimoto et al. 2014 Methods Mol. Biol. 1200, 579-606 (doi:10.1007/978-1-4939-1292-6_46)). This fact suggests that new lectins will be discovered either by a conventional screening approach or just by chance. It is also expected that new lectin domains including those found in enzymes as carbohydrate-binding modules will be generated in the future through evolution, although this has never been attempted on an experimental level. Based on the current state of the art, various methods of lectin engineering are available, by which lectin specificity and/or stability of a known lectin scaffold can be improved. However, the above observation implies that any protein scaffold, including those that have never been described as lectins, may be modified to acquire a sugar-binding function. In this review, possible approaches to confer sugar-binding properties on synthetic proteins and peptides are described.

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. Moreover, TIP60 forms larger structures upon the addition of a cationic protein. Therefore, TIP60 can be used as a modifiable nano-building block for further molecular assembly.

Self-Assembling Supramolecular Nanostructures Constructed from de Novo Extender Protein Nanobuilding Blocks.

The design of novel proteins that self-assemble into supramolecular complexes is important for development in nanobiotechnology and synthetic biology. Recently, we designed and created a protein nanobuilding block (PN-Block), WA20-foldon, by fusing an intermolecularly folded dimeric de novo WA20 protein and a trimeric foldon domain of T4 phage fibritin (Kobayashi et al., J. Am. Chem. Soc. 2015, 137, 11285). WA20-foldon formed several types of self-assembling nanoarchitectures in multiples of 6-mers, including a barrel-like hexamer and a tetrahedron-like dodecamer. In this study, to construct chain-like polymeric nanostructures, we designed de novo extender protein nanobuilding blocks (ePN-Blocks) by tandemly fusing two de novo binary-patterned WA20 proteins with various linkers. The ePN-Blocks with long helical linkers or flexible linkers were expressed in soluble fractions of Escherichia coli, and the purified ePN-Blocks were analyzed by native PAGE, size exclusion chromatography-multiangle light scattering (SEC-MALS), small-angle X-ray scattering (SAXS), and transmission electron microscopy. These results suggest formation of various structural homo-oligomers. Subsequently, we reconstructed hetero-oligomeric complexes from extender and stopper PN-Blocks by denaturation and refolding. The present SEC-MALS and SAXS analyses show that extender and stopper PN-Block (esPN-Block) heterocomplexes formed different types of extended chain-like conformations depending on their linker types. Moreover, atomic force microscopy imaging in liquid suggests that the esPN-Block heterocomplexes with metal ions further self-assembled into supramolecular nanostructures on mica surfaces. Taken together, the present data demonstrate that the design and construction of self-assembling PN-Blocks using de novo proteins is a useful strategy for building polymeric nanoarchitectures of supramolecular protein complexes.