Accurate and Fast Prediction of Intrinsically Disordered Protein by Multiple Protein Language Models and Ensemble Learning.

Intrinsically disordered proteins (IDPs) play a vital role in various biological processes and have attracted increasing attention in the past few decades. Predicting IDPs from the primary structures of proteins offers a rapid and facile means of protein analysis without necessitating crystal structures. In particular, machine learning methods have demonstrated their potential in this field. Recently, protein language models (PLMs) are emerging as a promising approach to extracting essential information from protein sequences and have been employed in protein modeling to utilize their advantages of precision and efficiency. In this article, we developed a novel IDP prediction method named IDP-ELM to predict the intrinsically disordered regions (IDRs) as well as their functions including disordered flexible linkers and disordered protein binding. This method utilizes high-dimensional representations extracted from several state-of-the-art PLMs and predicts IDRs by ensemble learning based on bidirectional recurrent neural networks. The performance of the method was evaluated on two independent test data sets from CAID (critical assessment of protein intrinsic disorder prediction) and CAID2, indicating notable improvements in terms of area under the receiver operating characteristic (AUC), Matthew's correlation coefficient (MCC), and F1 score. Moreover, IDP-ELM requires solely protein sequences as inputs and does not entail a time-consuming process of protein profile generation, which is a prerequisite for most existing state-of-the-art methods, enabling an accurate, fast, and convenient tool for proteome-level analysis. The corresponding reproducible source code and model weights are available at https://github.com/xu-shi-jie/idp-elm.

Evolutionary Engineering of a Cp*Rh(III) Complex-Linked Artificial Metalloenzyme with a Chimeric ß-Barrel Protein Scaffold.

Evolutionary engineering of our previously reported Cp*Rh(III)-linked artificial metalloenzyme was performed based on a DNA recombination strategy to improve its catalytic activity toward C(sp2)-H bond functionalization. Improved scaffold design was achieved with α-helical cap domains of fatty acid binding protein (FABP) embedded within the ß-barrel structure of nitrobindin (NB) as a chimeric protein scaffold for the artificial metalloenzyme. After optimization of the amino acid sequence by directed evolution methodology, an engineered variant, designated NBHLH1(Y119A/G149P) with enhanced performance and enhanced stability was obtained. Additional rounds of metalloenzyme evolution provided a Cp*Rh(III)-linked NBHLH1(Y119A/G149P) variant with a >35-fold increase in catalytic efficiency (kcat/KM) for cycloaddition of oxime and alkyne. Kinetic studies and MD simulations revealed that aromatic amino acid residues in the confined active-site form a hydrophobic core which binds to aromatic substrates adjacent to the Cp*Rh(III) complex. The metalloenzyme engineering process based on this DNA recombination strategy will serve as a powerful method for extensive optimization of the active-sites of artificial metalloenzymes.

One-Step Preparation of Fe/N/C Single-Atom Catalysts Containing Fe-N4 Sites from an Iron Complex Precursor with 5,6,7,8-Tetraphenyl-1,12-Diazatriphenylene Ligands.

Fe/N/C single-atom catalysts containing Fe-Nx sites prepared by pyrolysis are promising cathode materials for fuel cells and metal-air batteries due to their high oxygen reduction reaction (ORR) activities. We have developed iron complexes containing N2- or N3-chelating coordination structures with preorganized aromatic rings in a 1,12-diazatriphenylene framework tethering bromo substituents as precursors to precisely construct Fe-N4 sites in an Fe/N/C catalyst. One-step pyrolysis of the iron complex with carbon black forms atomically dispersed Fe-N4 sites without iron aggregates. X-ray absorption spectroscopy (XAS) and electrochemical measurements revealed that the iron complex with N3-coordination is more effectively converted to Fe-N4 sites catalyzing ORR with a TOF value of 0.21 e site-1 s-1 at 0.8 V vs. RHE. This indicates that the formation of Fe-N4 sites is controlled by precise tuning of the chemical structure of the iron complex precursor.

Thermally Controlled Construction of Fe-Nx Active Sites on the Edge of a Graphene Nanoribbon for an Electrocatalytic Oxygen Reduction Reaction.

Pyrolytically prepared iron and nitrogen codoped carbon (Fe/N/C) catalysts are promising nonprecious metal electrocatalysts for the oxygen reduction reaction (ORR) in fuel cell applications. Fabrication of the Fe/N/C catalysts with Fe-Nx active sites having precise structures is now required. We developed a strategy for thermally controlled construction of the Fe-Nx structure in Fe/N/C catalysts by applying a bottom-up synthetic methodology based on a N-doped graphene nanoribbon (N-GNR). The preorganized aromatic rings within the precursors assist graphitization during generation of the N-GNR structure with iron-coordinating sites. The Fe/N/C catalyst prepared from the N-GNR precursor, iron ion, and the carbon support Vulcan XC-72R provides a high onset potential of 0.88 V (vs reversible hydrogen electrode (RHE)) and promotes efficient four-electron ORR. X-ray absorption fine structure (XAFS) and X-ray photoelectron spectroscopy (XPS) studies reveal that the N-GNR precursor induces the formation of iron-coordinating nitrogen species during pyrolysis. The details of the graphitization process of the precursor were further investigated by analyzing the precursors pyrolyzed at various temperatures using MgO particles as a sacrificial template, with the results indicating that the graphitized structure was obtained at 700 °C. The preorganized N-GNR precursors and its pyrolysis conditions for graphitization are found to be important factors for generation of the Fe-Nx active sites along with the N-GNR structure in high-performance Fe/N/C catalysts for the ORR.

Construction of a whole-cell biohybrid catalyst using a Cp*Rh(III)-dithiophosphate complex as a precursor of a metal cofactor.

A whole-cell biohybrid catalyst where a (pentamethylcyclopentadienyl)rhodium(III) (Cp*Rh(III)) complex was covalently incorporated into the cavity of nitrobindin (NB), a ß-barrel protein, was prepared on an E. coli cell surface to produce isoquinolines via C(sp2)-H bond activation. In this whole-cell biohybrid system, the Cp*Rh(III)-dithiophosphate complex with latent catalytic activity was utilized as a precursor of the metal cofactor. Strong chelation of the dithiophosphate ligands protects the rhodium complex from being deactivated by abundant nucleophiles in cellular environments during conjugation of the cofactor with the protein scaffold. The whole-cell biohybrid catalyst was then activated upon addition of Ag+ ion to dissociate the dithiophosphate ligands and promoted cycloaddition of acetophenone oxime with diphenylacetylene. Furthermore, the activity of the Cp*Rh(III)-linked whole-cell biohybrid catalyst was enhanced 2.1-fold by introducing glutamate residues at positions adjacent to the Cp*Rh(III) cofactor. These results indicate that the use of the Cp*Rh(III)-dithiophosphate complex with switchable activity from a "latent" form to an "active" form provides a new strategy for generating whole-cell biohybrid catalysts.

Directed Evolution of a Cp*RhIII -Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification.

Directed evolution of Cp*RhIII -linked nitrobindin (NB), a biohybrid catalyst, was performed based on an in vitro screening approach. A key aspect of this effort was the establishment of a high-throughput screening (HTS) platform that involves an affinity purification step employing a starch-agarose resin for a maltose binding protein (MBP) tag. The HTS platform enables efficient preparation of the purified MBP-tagged biohybrid catalysts in a 96-well format and eliminates background influence of the host E. coli cells. Three rounds of directed evolution and screening of more than 4000 clones yielded a Cp*RhIII -linked NB(T98H/L100K/K127E) variant with a 4.9-fold enhanced activity for the cycloaddition of acetophenone oximes with alkynes. It is confirmed that this HTS platform for directed evolution provides an efficient strategy for generating highly active biohybrid catalysts incorporating a synthetic metal cofactor.

Incorporation of a Cp*Rh(III)-dithiophosphate Cofactor with Latent Activity into a Protein Scaffold Generates a Biohybrid Catalyst Promoting C(sp2)-H Bond Functionalization.

A Cp*Rh(III)-dithiophosphate cofactor with "latent" catalytic activity was developed to construct an artificial metalloenzyme representing a new type of biohybrid catalyst which is capable of promoting C(sp2)-H bond functionalization within the ß-barrel structure of nitrobindin (NB). To covalently conjugate the Cp*Rh(III) cofactor into a specific position of the hydrophobic cavity of NB via a maleimide-Cys linkage, strong chelation of the dithiophosphate ligand is employed to protect the rhodium metal center against attack by nucleophilic amino acid residues in the protein. It is found that subsequent addition of the Ag+ ion induces dissociation of the dithiophosphate ligands, thereby activating the catalytic activity of the Cp*Rh(III) cofactor. The resulting "active" biohybrid catalyst promotes cycloaddition of acetophenone oxime with diphenylacetylene via C(sp2)-H bond activation. This catalytic activity is enhanced 2.3-fold with the introduction of two glutamate residues (A100E/L125E) adjacent to the Cp*Rh(III) cofactor. The Cp*Rh(III) cofactor with switchable activity from a "latent" form to an "active" form provides a new strategy for generating biohybrid catalysts incorporating a variety of highly reactive transition metal complexes specifically within its protein scaffolds.

Triazolecarbaldehyde Reagents for One-Step N-Terminal Protein Modification.

Site-specific modification of peptides and proteins is a key aspect of protein engineering. We developed a method for modification of the N terminus of proteins using 1H-1,2,3-triazole-4-carbaldehyde (TA4C) derivatives, which can be prepared in one step. The N-terminal specific labeling of bioactive peptides and proteins with the TA4C derivatives proceeds under mild reaction conditions in excellent conversion (angiotensin I: 92 %, ribonuclease A: 90 %). This method enables site-specific conjugation of various functional molecules such as fluorophores, biotin, and polyethylene glycol attached to the triazole ring to the N terminus. Furthermore, a functional molecule modified with a primary amine moiety can be directly converted into a TA4C derivative through a Dimroth rearrangement reaction with 1-(4-nitrophenyl)-1H-1,2,3-triazole-4-carbaldehyde. This method can be used to obtain N-terminal-modified proteins via only two steps: 1) convenient preparation of a TA4C derivative with a functional group and 2) modification of the N terminus of the protein with the TA4C derivative.

Site-Specific Modification of Proteins through N-Terminal Azide Labeling and a Chelation-Assisted CuAAC Reaction.

Site-specific modification of peptides and proteins is an important method for introducing an artificial function to the protein surface. Recently, we found that new bioconjugation reagents, 6-(azidomethyl)-2-pyridinecarbaldehyde (6AMPC) derivatives, allow specific N-terminal modification and enhance the reaction rate of the subsequent bioconjugation in a chelation-assisted CuAAC reaction. The N-terminal specific azide-labeling of bioactive peptides and proteins occurs under mild reaction conditions with 6AMPC derivatives (angiotensin I: 90%, ribonuclease A: 90%). Kinetic analysis of the CuAAC reaction with azide-labeled proteins reveals that the ligation is promoted in the presence of a copper-chelating pyridine moiety. Importantly, the introduction of an electron-donating methoxy group to the pyridine moiety further accelerates the CuAAC ligation. We demonstrate that this method enables site-specific conjugation of various functional molecules such as fluorophores, biotin, and polyethylene glycol.

Hemoproteins Reconstituted with Artificial Metal Complexes as Biohybrid Catalysts.

In nature, heme cofactor-containing proteins participate not only in electron transfer and O2 storage and transport but also in biosynthesis and degradation. The simplest and representative cofactor, heme b, is bound within the heme pocket via noncovalent interaction in many hemoproteins, suggesting that the cofactor is removable from the protein, leaving a unique cavity. Since the cavity functions as a coordination sphere for heme, it is of particular interest to investigate replacement of native heme with an artificial metal complex, because the substituted metal complex will be stabilized in the heme pocket while providing alternative chemical properties. Thus, cofactor substitution has great potential for engineering of hemoproteins with alternative functions. For these studies, myoglobin has been a focus of our investigations, because it is a well-known oxygen storage hemoprotein. However, the heme pocket of myoglobin has been only arranged for stabilizing the heme-bound dioxygen, so the structure is not suitable for activation of small molecules such as H2O2 and O2 as well as for binding an external substrate. Thus, the conversion of myoglobin to an enzyme-like biocatalyst has presented significant challenges. The results of our investigations have provided useful information for chemists and biologists. Our own efforts to develop functionalized myoglobin have focused on the incorporation of a chemically modified cofactor into apomyoglobin in order to (1) construct an artificial substrate-binding site near the heme pocket, (2) increase cofactor reactivity, or (3) promote a new reaction that has never before been catalyzed by a native heme enzyme. In pursuing these objectives, we first found that myoglobin reconstituted with heme having a chemically modified heme-propionate side chain at the exit of the heme pocket has peroxidase activity with respect to oxidation of phenol derivatives. Our recent investigations have succeeded in enhancing oxidation and oxygenation activities of myoglobin as well as promoting new reactions by reconstitution of myoglobin with new porphyrinoid metal complexes. Incorporation of suitable metal porphyrinoids into the heme pocket has produced artificial enzymes capable of efficiently generating reactive high valent metal-oxo and metallocarbene intermediates to achieve the catalytic hydroxylation of C(sp3)-H bonds and cyclopropanation of olefin molecules, respectively. In other efforts, we have focused on nitrobindin, an NO-binding hemoprotein, because aponitrobindin includes a ß-barrel cavity, which provides a robust structure highly similar to that of the native holoprotein. It was expected that the aponitrobindin would be suitable for development as a protein scaffold for a metal complex. Recently, it was confirmed that several organometallic complexes can bind to this scaffold and function as catalysts promoting hydrogen evolution or C-C bond formation. The hydrophobic ß-barrel structure plays a significant role in substrate binding as well as controlling the stereoselectivity of the reactions. Furthermore, these catalytic activities and stereoselectivities are remarkably improved by mutation-dependent modifications of the cavity structure for the artificial cofactor. This Account demonstrates how apoproteins of hemoproteins can provide useful protein scaffolds for metal complexes. Further development of these concepts will provide a useful strategy for generation of robust and useful artificial metalloenzymes.

A water-soluble supramolecular complex that mimics the heme/copper hetero-binuclear site of cytochrome c oxidase.

In mitochondria, cytochrome c oxidase (CcO) catalyses the reduction of oxygen (O2) to water by using a heme/copper hetero-binuclear active site. Here we report a highly efficient supramolecular approach for the construction of a water-soluble biomimetic model for the active site of CcO. A tridentate copper(ii) complex was fixed onto 5,10,15,20-tetrakis(4-sulfonatophenyl)porphinatoiron(iii) (FeIIITPPS) through supramolecular complexation between FeIIITPPS and a per-O-methylated ß-cyclodextrin dimer linked by a (2,2':6',2''-terpyridyl)copper(ii) complex (CuIITerpyCD2). The reduced FeIITPPS/CuITerpyCD2 complex reacted with O2 in an aqueous solution at pH 7 and 25 °C to form a superoxo-type FeIII-O2-/CuI complex in a manner similar to CcO. The pH-dependent autoxidation of the O2 complex suggests that water molecules gathered at the distal Cu site are possibly involved in the FeIII-O2-/CuI superoxo complex in an aqueous solution. Electrochemical analysis using a rotating disk electrode demonstrated the role of the FeTPPS/CuTerpyCD2 hetero-binuclear structure in the catalytic O2 reduction reaction.

Mitochondria-Targeting Polyamine-Protoporphyrin Conjugates for Photodynamic Therapy.

Two polyamine derivatives of protoporphyrin IX (PPIX) were tested as photodynamic therapy (PDT) agents in HT29 colorectal cancer and HEP3B liver cancer cell lines. These compounds exhibit excellent singlet oxygen quantum yields and show strong in vitro PDT efficacy after 660 nm laser irradiation, whereas exogenous PPIX itself exhibits much weaker PDT effects. Confocal microscopy imaging studies reveal that a protoporphyrin derivative with eight amine moieties has excellent water solubility, and localizes mainly in the mitochondria of both HT29 and HEP3B cells, whereas the cellular distribution of a protoporphyrin derivative with four amine moieties is not as specific. This work demonstrates that polyamine moieties on macrocycles can enhance PDT efficacy by targeting mitochondria.

Bimetallic M/N/C catalysts prepared from π-expanded metal salen precursors toward an efficient oxygen reduction reaction.

Nonprecious metal electrocatalysts are being explored as alternatives to platinum-group metal electrocatalysts for the oxygen reduction reaction (ORR) which is required for cathode materials in fuel cells. Herein, we describe a new method for preparing bimetallic nitrogen-containing carbon catalysts with high ORR activity using π-expanded M(salen) precursors. The M/N/C and bimetallic FeM/N/C ORR catalysts were obtained by pyrolysis of a mixture of a carbon support (Vulcan XC-72R) and the metal complex as a precursor. The bimetallic FeCu catalyst prepared from Fe and Cu complexes with the N,N'-bis(2-hydroxy-1-naphthylidene)-1,2-phenylenediamine ligand (2NAPD) is found to have an onset potential of 0.87 V, which is positively shifted by 50 mV from that of the catalyst prepared from the monometallic Fe(2NAPD) complex. The FeCu/N/C catalyst promotes efficient four-electron reduction in the ORR. High-resolution transmission electron microscopy studies reveal that both Fe and Cu metals together with pyridinic nitrogen species are highly dispersed within the carbonaceous structure in FeCu/2NAPD@VC, suggesting that the N-coordinated Fe and Cu sites promote efficient four-electron reduction of O2. This new methodology facilitates design of nonprecious bimetallic carbon catalysts with excellent ORR activity.

Interdomain flip-flop motion visualized in flavocytochrome cellobiose dehydrogenase using high-speed atomic force microscopy during catalysis.

Cellobiose dehydrogenase (CDH) is a dual domain flavocytochrome, which consists of a dehydrogenase (DH) domain containing a flavin adenine dinucleotide and a cytochrome (CYT) domain containing b-type heme. To directly visualize the dynamic domain motion of class-I CDH from Phanerochaete chrysosporium (PcCDH) during catalysis using high-speed atomic force microscopy, the apo-form of PcCDH was anchored to a heme-immobilized flat gold surface that can specifically fix the orientation of the CYT domain. The two domains of CDH are found to be immobile in the absence of cellobiose, whereas the addition of cellobiose triggers an interdomain flip-flop motion involving domain-domain association and dissociation. Our results indicate that dynamic motion of a dual domain enzyme during catalysis induces efficient electron transfer to an external electron acceptor.

A Pyrene-Linked Cavity within a ß-Barrel Protein Promotes an Asymmetric Diels-Alder Reaction.

A unique π-expanded reaction cavity tethering a polycyclic moiety which provides a platform for substrate binding was constructed within the robust ß-barrel structure of nitrobindin (NB). NB variants with cavities of different sizes and shapes are coupled with N-(1-pyrenyl)maleimide (Pyr) to prepare a series of NB-Pyr conjugates. The orientation of the pyrene moiety is fixed within the cavity by the coupling reaction. The fluorescent quenching analysis of NB-Pyr indicates that azachalcone (aza), which is a dienophile for a Diels-Alder (DA) reaction, is efficiently incorporated within the pyrene-linked reaction cavity by the aromatic interaction. The DA reaction between aza and cyclopentadiene proceeds within the reaction cavity of NB-Pyr in the presence of CuII ion in high yield and high enantio- and regioselectivity.

A supramolecular assembly based on an engineered hemoprotein exhibiting a thermal stimulus-driven conversion to a new distinct supramolecular structure.

Supramolecular assembly of an engineered hemoprotein with an externally-attached heme moiety via an azobenzene or stilbene linker demonstrates drastic structural transitions between two distinct forms: the thermodynamically stable fiber-type assembly and the kinetically trapped metastable micelle-type assembly induced by transient thermal stimulus.

Artificial Diels-Alderase based on the transmembrane protein FhuA.

Copper(I) and copper(II) complexes were covalently linked to an engineered variant of the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA ΔCVF(tev)). Copper(I) was incorporated using an N-heterocyclic carbene (NHC) ligand equipped with a maleimide group on the side arm at the imidazole nitrogen. Copper(II) was attached by coordination to a terpyridyl ligand. The spacer length was varied in the back of the ligand framework. These biohybrid catalysts were shown to be active in the Diels-Alder reaction of a chalcone derivative with cyclopentadiene to preferentially give the endo product.

Photocatalytic Properties of TiO2 Composites Immobilized with Gold Nanoparticle Assemblies Using the Streptavidin-Biotin Interaction.

A method using biomolecules to precisely fabricate the morphology of metal nanoparticles immobilized on the surface of a semiconductor using biomolecules is described. A biotin moiety (Biot) is introduced onto the surface of a gold nanoparticle (AuNP) by covalent coupling with α-lipoic acid to assemble AuNPs in the presence of streptavidin (STV). The assembly of Biot-AuNP/STV is immobilized on the surface of TiO2 chemically modified with 1-(3-aminopropyl)silatrane (APS) to provide a positively charged surface. The Au content immobilized on the surface of TiO2 is clearly increased to 9.5 wt % (Au) as a result of the STV-biotin interaction and the electrostatic interaction between negatively charged Biot-AuNPs and the positively charged surface of APS/TiO2. Transmission electron microscopy (TEM) analysis reveals that the composite has an ordered surface geometry in which Biot-AuNPs are spread over the composite surface in two dimensions. The photocatalytic activity toward decomposition of methyl orange dye promoted by this composite is 55%, which is higher than that of the other composites. The Biot-AuNP/STV@APS/TiO2 composite efficiently reduces O2 molecules at Eonset = -0.23 V vs Ag|AgCl, which is more positive than that of other composites (Eonset = -0.40 to -0.32 V). The result suggests that an increased number of AuNPs immobilized in close contact with the TiO2 surface facilitates photoinduced charge transfer. This strategy, which takes advantage of the specific interactions provided by biomolecules and the chemical modification on the surface, has remarkable potential for efficient fabrication of metal nanoparticles on the surface of the semiconductor, which accelerates the reduction of oxygen molecules.

Oxygen-binding Protein Fiber and Microgel: Supramolecular Myoglobin-Poly(acrylate) Conjugates.

A supramolecular conjugate of myoglobin (Mb) and water-soluble poly(acrylate), (PA5k and PA25k , where 5k and 25k represent the molecular weight of the polymers, respectively), is constructed on the basis of a noncovalent heme-heme pocket interaction. The modified heme with an amino group linked to the terminus of one of the heme-propionates is coupled to the side-chain carboxyl groups of poly(acrylate) activated by N-hydroxysuccinimide. The ratios of the heme-modified monomer unit and the unmodified monomer unit (m:n) in the polymer chains of Heme-PA5k and Heme-PA25k were determined to be 4.5:95.5 and 3.1:96.9, respectively. Subsequent addition of apoMb to the conjugated polymers provides Mb-connected fibrous nanostructures confirmed by atomic force microscopy. A mixture of the heme-modified polymer and dimeric apomyoglobin as a cross-linker forms a microgel in which the reconstituted myoglobin retains its native exogenous ligand binding activity.

Construction of a hybrid biocatalyst containing a covalently-linked terpyridine metal complex within a cavity of aponitrobindin.

A hybrid biocatalyst containing a metal terpyridine (tpy) complex within a rigid ß-barrel protein nitrobindin (NB) is constructed. A tpy ligand with a maleimide group, N-[2-([2,2':6',2''-terpyridin]-4'-yloxy)ethyl]maleimide (1), was covalently linked to Cys96 inside the cavity of NB to prepare a conjugate NB-1. Binding of Cu(2+), Zn(2+), or Co(2+) ion to the tpy ligand in NB-1 was confirmed by UV-vis spectroscopy and ESI-TOF MS measurements. Cu(2+)-bound NB-1 is found to catalyze a Diels-Alder reaction between azachalcone and cyclopentadiene in 22% yield, which is higher than that of the Cu(2+)-tpy complex without the NB matrix. The results suggest that the hydrophobic cavity close to the copper active site within the NB scaffold supports the binding of the two substrates, dienophile and diene, to promote the reaction.