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.

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.

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.

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.

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.

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.

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.

Functional characterization of FABP3, 5 and 7 gene variants identified in schizophrenia and autism spectrum disorder and mouse behavioral studies.

Disturbances of lipid metabolism have been implicated in psychiatric illnesses. We previously reported an association between the gene for fatty acid binding protein 7 (FABP7) and schizophrenia. Furthermore, we identified and reported several rare non-synonymous polymorphisms of the brain-expressed genes FABP3, FABP5 and FABP7 from schizophrenia and autism spectrum disorder (ASD), diseases known to part share genetic architecture. Here, we conducted further studies to better understand the contribution these genes make to the pathogenesis of schizophrenia and ASD. In postmortem brains, we detected altered mRNA expression levels of FABP5 in schizophrenia, and of FABP7 in ASD and altered FABP5 in peripheral lymphocytes. Using a patient cohort, comprehensive mutation screening identified six missense and two frameshift variants from the three FABP genes. The two frameshift proteins, FABP3 E132fs and FABP7 N80fs, formed cellular aggregates and were unstable when expressed in cultured cells. The four missense mutants with predicted possible damaging outcomes showed no changes in intracellular localization. Examining ligand binding properties, FABP7 S86G and FABP7 V126L lost their preference for docosahexaenoic acid to linoleic acid. Finally, mice deficient in Fabp3, Fabp5 and Fabp7 were evaluated in a systematic behavioral test battery. The Fabp3 knockout (KO) mice showed decreased social memory and novelty seeking, and Fabp7 KO mice displayed hyperactive and anxiety-related phenotypes, while Fabp5 KO mice showed no apparent phenotypes. In conclusion, disturbances in brain-expressed FABPs could represent an underlying disease mechanism in a proportion of schizophrenia and ASD sufferers.

Characterization of silk gland ribosomes from a bivoltine caddisfly, Stenopsyche marmorata: translational suppression of a silk protein in cold conditions.

Larval Stenopsyche marmorata constructs food capture nets and fixed retreats underwater using self-produced proteinaceous silk fibers. In the Chikuma River (Nagano Prefecture, Japan) S. marmorata has a bivoltine life cycle; overwintering larvae grow slowly with reduced net spinning activity in winter. We recently reported constant transcript abundance of S. marmorata silk protein 1 (Smsp-1), a core S. marmorata silk fiber component, in all seasons, implying translational suppression in the silk gland during winter. Herein, we prepared and characterized silk gland ribosomes from seasonally collected S. marmorata larvae. Ribosomes from silk glands immediately frozen in liquid nitrogen (LN2) after dissection exhibited comparable translation elongation activity in spring, summer, and autumn. Conversely, silk glands obtained in winter did not contain active ribosomes and Smsp-1. Ribosomes from silk glands immersed in ice-cold physiological saline solution for approximately 4 h were translationally inactive, despite summer collection and Smsp-1 expression. The ribosomal inactivation occurs because of defects in the formation of 80S ribosomes, presumably due to splitting of 60S subunits containing 28S rRNA with central hidden break, in response to cold stress. These results suggest a novel-type ribosome-regulated translation control mechanism.

Self-Assembling Nano-Architectures Created from a Protein Nano-Building Block Using an Intermolecularly Folded Dimeric de Novo Protein.

The design of novel proteins that self-assemble into supramolecular complexes is an important step in the development of synthetic biology and nanotechnology. Recently, we described the three-dimensional structure of WA20, a de novo protein that forms an intermolecularly folded dimeric 4-helix bundle (PDB code 3VJF ). To harness the unusual intertwined structure of WA20 for the self-assembly of supramolecular nanostructures, we created a protein nanobuilding block (PN-Block), called WA20-foldon, by fusing the dimeric structure of WA20 to the trimeric foldon domain of fibritin from bacteriophage T4. The WA20-foldon fusion protein was expressed in the soluble fraction in Escherichia coli, purified, and shown to form several homooligomeric forms. The stable oligomeric forms were further purified and characterized by a range of biophysical techniques. Size exclusion chromatography, multiangle light scattering, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS) analyses indicate that the small (S form), middle (M form), and large (L form) forms of the WA20-foldon oligomers exist as hexamer (6-mer), dodecamer (12-mer), and octadecamer (18-mer), respectively. These findings suggest that the oligomers in multiples of 6-mer are stably formed by fusing the interdigitated dimer of WA20 with the trimer of foldon domain. Pair-distance distribution functions obtained from the Fourier inversion of the SAXS data suggest that the S and M forms have barrel- and tetrahedron-like shapes, respectively. These results demonstrate that the de novo WA20-foldon is an effective building block for the creation of self-assembling artificial nanoarchitectures.

Molecular cloning, gene expression analysis, and recombinant protein expression of novel silk proteins from larvae of a retreat-maker caddisfly, Stenopsyche marmorata.

Retreat-maker larvae of Stenopsyche marmorata, one of the major caddisfly species in Japan, produce silk threads and adhesives to build food capture nets and protective nests in water. Research on these underwater adhesive silk proteins potentially leads to the development of new functional biofiber materials. Recently, we identified four major S. marmorata silk proteins (Smsps), Smsp-1, Smsp-2, Smsp-3, and Smsp-4 from silk glands of S. marmorata larvae. In this study, we cloned full-length cDNAs of Smsp-2, Smsp-3, and Smsp-4 from the cDNA library of the S. marmorata silk glands to reveal the primary sequences of Smsps. Homology search results of the deduced amino acid sequences indicate that Smsp-2 and Smsp-4 are novel proteins. The Smsp-2 sequence [167 amino acids (aa)] has an array of GYD-rich repeat motifs and two (SX)4E motifs. The Smsp-4 sequence (132 aa) contains a number of GW-rich repeat motifs and three (SX)4E motifs. The Smsp-3 sequence (248 aa) exhibits high homology with fibroin light chain of other caddisflies. Gene expression analysis of Smsps by real-time PCR suggested that the gene expression of Smsp-1 and Smsp-3 was relatively stable throughout the year, whereas that of Smsp-2 and Smsp-4 varied seasonally. Furthermore, Smsps recombinant protein expression was successfully performed in Escherichia coli. The study provides new molecular insights into caddisfly aquatic silk and its potential for future applications.

Phylogenetic relationships of bitterling fishes (Teleostei: Cypriniformes: Acheilognathinae), inferred from mitochondrial cytochrome B sequences.

Bitterling (Teleostei: Acheilognathinae) are small cyprinid fishes with a discrete distribution in East Asia and Europe. We used a complete mitochondrial cytochrome b sequence (1141 bp) from 49 species or subspecies in three genera (Tanakia, Rhodeus, and Acheilognathus), sampled across the major part of their distribution, to elucidate their phylogeny and biogeography, focusing particularly on their origin and dispersal. Based on high support value, the monophyletic Acheilognathinae separated into two major clades, Acheilognathus and Tanakia-Rhodeus. In the latter clade, the monophyly of Rhodeus was poorly supported, though it was topologically nested in Tanakia. On the basis of molecular-clock calibration, both clades diverged in the middle Miocene, with Tanakia-Rhodeus diverging slightly earlier than Acheilognathus. The Tanakia-Rhodeus clade expanded its distribution westward from the Far East, eventually reaching Europe, while Acheilognathus dispersed in the temperate regions of East Asia. A feature common to both clades is that most extant species, including Japanese endemics, appeared by the end of the Pliocene, corresponding with the present delineation of the Japanese archipelago. Autumn-spawning species with an embryonic diapause, unique to bitterling among cyprinid fishes, formed two distinct lineages (barbatulusrhombeus and longipinnis-typus) within Acheilognathus. The estimated time of divergence of the two lineages was approximately from the late Pliocene, a period characterized by glaciations. The timing of divergence suggests that the shift of spawning from spring to autumn, coupled with embryonic diapause, convergently emerged twice in the evolution of bitterling, possibly as an adaptation to the climate of the late Pliocene.

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

Rhodeus albomarginatus, new species, is described from the Lvjiang River, a tributary flowing into Poyang Lake of Yangtze River basin, in Anhui Province, China. It is distinguished from all congeneric species by unique combination of characters: branched dorsal-fin rays 10; branched anal-fin rays 10-11; longest simple rays of dorsal and anal fins strong and stiff, distally segmented; pelvic fin rays i 6; longitudinal scale series 34-36; transverse scale series 11; pored scales 4-7; vertebrae 33-34; colour pattern of adult males (iris black, belly reddish-orange, central part of caudal fin red, dorsal and anal fins of males edged with white margin).

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.

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.

Solution structure of IseA, an inhibitor protein of DL-endopeptidases from Bacillus subtilis, reveals a novel fold with a characteristic inhibitory loop.

In Bacillus subtilis, LytE, LytF, CwlS, and CwlO are vegetative autolysins, DL-endopeptidases in the NlpC/P60 family, and play essential roles in cell growth and separation. IseA (YoeB) is a proteinaceous inhibitor against the DL-endopeptidases, peptidoglycan hydrolases. Overexpression of IseA caused significantly long chained cell morphology, because IseA inhibits the cell separation DL-endopeptidases post-translationally. Here, we report the first three-dimensional structure of IseA, determined by NMR spectroscopy. The structure includes a single domain consisting of three α-helices, one 3(10)-helix, and eight ß-strands, which is a novel fold like a "hacksaw." Noteworthy is a dynamic loop between ß4 and the 3(10)-helix, which resembles a "blade." The electrostatic potential distribution shows that most of the surface is positively charged, but the region around the loop is negatively charged. In contrast, the LytF active-site cleft is expected to be positively charged. NMR chemical shift perturbation of IseA interacting with LytF indicated that potential interaction sites are located around the loop. Furthermore, the IseA mutants D100K/D102K and G99P/G101P at the loop showed dramatic loss of inhibition activity against LytF, compared with wild-type IseA, indicating that the ß4-3(10) loop plays an important role in inhibition. Moreover, we built a complex structure model of IseA-LytF by docking simulation, suggesting that the ß4-3(10) loop of IseA gets stuck deep in the cleft of LytF, and the active site is occluded. These results suggest a novel inhibition mechanism of the hacksaw-like structure, which is different from known inhibitor proteins, through interactions around the characteristic loop regions with the active-site cleft of enzymes.

Long-range periodic sequence of the cement/silk protein of Stenopsyche marmorata: purification and biochemical characterisation.

The long-range periodic amino acid sequence of the bifunctional silk/cement protein from larvae of the caddisfly, Stenopsyche marmorata, is discussed in this study. The protein, named the S. marmorata silk protein (Smsp-1), was first purified to electrophoretic homogeneity. The results of Edman-based sequencing of Smsp-1 tryptic digests were consistent with the amino acid sequence deduced from a cDNA clone of the Smsp-1 gene. All undetected amino acids in the Edman-based sequencing were encoded as Ser, suggesting the presence of O-phospho-Ser. (31)P-NMR and an O-phospho-amino acid analysis successfully showed that the O-phospho-Ser residue occurred in a clustered manner, serving a cement function for Smsp-1. Two patterns of non-phosphorylated repeats, -SLGPYGDPRGDXLGPYGG- (X = V, G or D) and -GVGPYGDGLGPYGG-, were enriched in Smsp-1 compared with the O-phospho-Ser cluster, and have fibre-forming functions.

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.