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

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

Evolutionary Engineering of a Cp*Rh(III) Complex-Linked Artificial Metalloenzyme with a Chimeric β‑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 β-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

Alignment of Gold Clusters on DNA via a DNA-Recognizing Zinc Finger-Metallothionein Fusion Protein

The complementary recognition of base pairs (bp) is the major strategy in the “DNA lithography” of gold (Au) clusters or nanoparticles, where single-stranded DNAs sulfurized at their termini are generally used to bind Au clusters or nanoparticles. In this report, we discuss a new material that can be used to locate Au clusters on the desired positions of DNA. For this purpose, we combined a two-domain zinc finger (ZF) and the analogue of α domain of rat’s liver metallothionein (MT) to utilize the DNA-recognizing ability of ZF motifs and the heavy-metal binding ability of MTs, and prepared an artificial fusion protein, ZFZF-MTα (1). Titration experiments monitored by absorption and circular dichroism spectroscopies, as well as an electrophoretic mobility shift assay and quantification using 5,5′-dithiobis(2-nitrobenzoic acid), clarified that (1) the ZF domain traps two divalent metal ions to fold in a ZF structure with M(Cys)2(His)2 (M = Co, Zn, and Cd) coordination units, (2) the MT domain traps metal ions to form clusters (Cd2+ particularly forms a Cd4(Cys)9 cluster) without interfering with the folding of the ZF domain, and (3) 1 recognizes the 5′-GGGGGG-3′ (G6) bp sequence in the presence of Zn2+ based on the amino acid sequence encoded in the ZF domain. The titration of Au11(PPh3)8Cl3 (Au11) into the solution of 1 in the presence of Zn2+ revealed that the MT domain strongly binds the Au11 cluster with a 1:1 ratio, and a Au11-containing conjugate, ZF(Zn)ZF(Zn)-MTα(Au11) was obtained. Transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy showed that this conjugate maintains an Au11 core in the form of Au11(PPh3)4(S-Cys)6 without disturbing the folding nature of the ZF domain. ZF(Zn)ZF(Zn)-MTα(Au11) recognizes the bp sequence G6 with Kd1 = 450 nM, while simultaneously forming a dimer on the DNA with Kd2 = 200 nM. TEM experiments showed that the conjugates form parallelograms or triangles (defective parallelograms) on a double crossover (dx) DNA, according to the positions of G6 encoded in the dx DNA

Crystal Structure and Spectroscopic Studies of a Stable Mixed-Valent State of the Hemerythrin-like Domain of a Bacterial Chemotaxis Protein

The bacterial chemotaxis protein of Desulfovibrio vulgaris DcrH (DcrH-Hr) functions as an O2-sensing protein. This protein has a hemerythrin-like domain that includes a nonheme diiron center analogous to the diiron center of the hemerythrin (Hr) family. Interestingly, the O2 affinity of DcrH-Hr is 3.3 × 106 M–1, a value 25-fold higher than that of the Pectinaria gouldii Hr. This high affinity arises from the fast association of the O2 ligand with DcrH-Hr (kon = 5.3 × 108 M–1 s–1), which is made possible by a hydrophobic tunnel that accelerates the passage of the O2 ligand to the diiron site. Furthermore, the autoxidation kinetics indicate that the rate of autoxidation of DcrH-Hr is 54-fold higher than that of P. gouldii Hr, indicating that the oxy form of DcrH-Hr is not stable toward autoxidation. More importantly, a mixed-valent state, semimetR, which was spectroscopically observed in previous Hr studies, was found to be stable for over 1 week and isolable in the case of DcrH-Hr. The high-resolution crystal structures of the semimetR- (1.8 Å) and met-DcrH-Hr (1.4 Å) indicate that the semimetR- and met-DcrH-Hr species have very similar coordination geometry at the diiron site

Calcium Ion Responsive DNA Binding in a Zinc Finger Fusion Protein

Zinc finger fusion proteins, having a Ca-binding site from troponin C, were created to develop Ca-responsive regulation of DNA binding. The typical zinc finger folding of a novel fusion protein with a single finger, F2-Tn, was investigated using UV−vis spectroscopy of the Co-substituted form and CD experiments. Detailed structural analyses of F2-Tn/Zn2+ using NMR experiments and structural calculations clarify that our fusion protein gives a native zinc finger folding with the artificial Ca-binding domain intervening two helices. The Ca-responsive DNA-binding affinity of troponin-fused protein with two fingers (using F1F2-Tn) was investigated by electrophoretic mobility shift assay (EMSA). EMSA analyses of F1F2-Tn were performed under the conditions of various concentrations of the Ca ion. F1F2-Tn has a Kd value of 5.8 nM in the absence of Ca ion and shows a higher Kd value of 13 nM in the presence of 100 equiv of Ca ion. The artificially designed fusion zinc finger protein with a Ca-binding domain has Ca-responsive DNA-binding affinity. It is leading to a better understanding of the construction of zinc finger-based artificial transcriptional factors with a Ca switch

Photoinduced Hydrogen Evolution Catalyzed by a Synthetic Diiron Dithiolate Complex Embedded within a Protein Matrix

The hydrogen-evolving diiron complex, (μ-S)₂Fe₂(CO)₆ with a tethered maleimide moiety was synthesized and covalently embedded within the cavity of a rigid β-barrel protein matrix by coupling a maleimide moiety to a cysteine residue within the β-barrel. The (μ-S)₂Fe₂(CO)₆ core within the cavity was characterized by UV–vis absorption and a characteristic CO vibration determined by IR measurements. The diiron complex embedded within the cavity retains the necessary catalytic activity (TON up to 130 for 6 h) to evolve H₂ via a photocatalytic cycle with a Ru photosensitizer in a solution of 100 mM ascorbate and 50 mM Tris/HCl at pH 4.0 and 25 °C

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

Mononuclear Ca(II)−Bulky Aryl−Phosphate Monoanion and Dianion Complexes with Ortho-Amide Groups

Two new mononuclear Ca(II) complexes with aryl dihydrogen phosphate ligands having two strategically oriented bulky amide groups, 2,6-(Ph3CCONH)2C6H3OPO3H2 (1), including one with a phosphate monoanion, (NMe4)[CaII{O2P(OH)OC6H3-2,6-(NHCOCPh3)2}3(N⋮CMe)3] (3), and one with a phosphate dianion, [CaII{O3POC6H3-2,6-(NHCOCPh3)2}(H2O)3(MeOH)2] (4). Both are analogues for the NH···O hydrogen bonds in the active site of Ca(II)-containing phosphotransferase. Crystallographic studies of these Ca(II) complexes revealed that the amide NHs are directed to uncoordinated O atoms of the phosphates, and the IR and 1H NMR spectra indicate that strong NH···O hydrogen bonds are formed only in the phosphate dianion state. The ligand exchange reaction of 3 with a non-hydrogen-bonded phosphate ligand shows that the NH···O hydrogen bonds prevent the Ca−O bond from dissociation. A scatter plot analysis comparing the distance of a Ca−O bond with the Ca−O−P angle, the Fourier density analysis, and DFT calculations reveal that a partial degree of covalency in the Ca−O(phosphate) bonds is present

Dinuclear Calcium Complexes with Intramolecularly NH···O Hydrogen-Bonded Dicarboxylate Ligands

A novel dinuclear calcium complex, [Ca2{(2-OCO-3-CH3C6H3NHCO)2C(CH3)2}2(CH3OH)6] (1), was synthesized as a structural model of 8-coordinated Ca(II) ions in the double calcium-binding site of thermolysin. The complex has four NH···O hydrogen bonds between the amide NH and the carboxylate oxygen anion. Two types of bridging coordination of the carboxylate ligand to Ca(II) were found in 1. The amide NH forms a strong NH···O hydrogen bond with the anionic oxygen of the two carboxylate oxygens. A ligand-exchange reaction between the dinuclear calcium complex and eight equimolar amounts of 2,4,6-trimethylbenzoic acid or 2-CH3-6-t-BuCONHC6H3COOH indicates that the NH···O hydrogen bond prevents the dissociation of the Ca−O bond