2D Atomic-Molecular Heterojunctions toward Brainoid Applications.

Brainoid computing using 2D atomic crystals and their heterostructures, by emulating the human brain's remarkable efficiency and minimal energy consumption in information processing, poses a formidable solution to the energy-efficiency and processing speed constraints inherent in the von Neumann architecture. However, conventional 2D material based heterostructures employed in brainoid devices are beset with limitations, performance uniformity, fabrication intricacies, and weak interfacial adhesion, which restrain their broader application. The introduction of novel 2D atomic-molecular heterojunctions (2DAMH), achieved through covalent functionalization of 2D materials with functional molecules, ushers in a new era for brain-like devices by providing both stability and tunability of functionalities. This review chiefly delves into the electronic attributes of 2DAMH derived from the synergy of polymer materials with 2D materials, emphasizing the most recent advancements in their utilization within memristive devices, particularly their potential in replicating the functionality of biological synapses. Despite ongoing challenges pertaining to precision in modification, scalability in production, and the refinement of underlying theories, the proliferation of innovative research is actively pursuing solutions. These endeavors illuminate the vast potential for incorporating 2DAMH within brain-inspired intelligent systems, highlighting the prospect of achieving a more efficient and energy-conserving computing paradigm.

Allergenicity assessment of ß-lactoglobulin ferulic acid-glucose conjugates.

We prepared the ß-lactoglobulin (BLG)-ferulic acid (FA)-glucose (Glu) conjugates by alkaline method and Maillard reaction to assess the allergenicity. FA and Glu can form a ternary covalent conjugate with BLG, as evidenced by the shortening of SEC retention time, upward migration of SDS-PAGE protein bands, considerable decrease in free amino and sulfhydryl content, and changes in multistructure. BLG-Glu-FA conjugates weakly bound to immunoglobulin E in allergic sera was weak, reduced interleukin 4 and tumor necrosis factor α levels in RBL-2H3 cells and histamin and interleukin 6 secretion levels in KU812 cells, and inhibited the nuclear factor-κB signaling pathway. In vivo experiments showed that the conjugates regulated T-cell homeostasis in mouse splenic and mesenteric lymphocytes and attenuated splenic and duodenal immune injury. Therefore, the conjugates of BLG with FA combined with Glu altered the epitope structure and exhibited low allergenicity.

Preparation of 4-Amino-3-hydrazino-1,2,4-triazol-5-thiol-Modified Graphene Oxide and Its Greatly Enhanced Selective Adsorption of Gallium in Aqueous Solution.

Efficient recovery of gallium (Ga) from vanadium slag processing residue (VSPR) solution is of great significance for environmental protection and resource utilization, but improving its selective adsorption against the coexisting Sc3+ and In3+ is still challenging. Herein, a novel adsorbent consisting of 4-amino-3-hydrazino-1,2,4-triazol-5-thiol (AHTZT)-modified graphene oxide (GO-AHTZT) was successfully synthesized that exhibits a higher adsorption selectivity for Ga3+ in VSPR solution with coexisting Sc3+ and In3+. Under optimal conditions, the adsorption capacity of GO-AHTZT for Ga3+ can reach 23.92 mg g-1, which is 4.9 and 12.6 times higher than that for Sc3+ (4.87 mg g-1) and In3+ (1.90 mg g-1) adsorption, indicating the excellent anti-interference ability of GO-AHTZT against Sc3+ and In3+. The process and mechanism of Ga3+ adsorption onto GO-AHTZT was also studied and discussed in detail. By measuring the adsorption process and by characterizing the adsorbent before and after adsorption, we demonstrate that the selective interaction between the Ga3+- and N-containing groups in AHTZT is the main reason for the improved adsorption selectivity. This work opens up an avenue for the design and synthesis of highly selective adsorbents for Ga3+ in complex VSPR solutions.

Oligonucleotide-Based Photoaffinity Probes: Chemical Tools and Applications for Protein Labeling.

A variety of proteins interact with DNA and RNA, including polymerases, histones, ribosomes, transcription factors, and repair enzymes. However, the transient non-covalent nature of these interactions poses challenges for analysis. Introducing a covalent bond between proteins and DNA via photochemical activation of a photosensitive functional group introduced onto nucleic acids offers a means to stabilize these often weak interactions without significantly altering the binding interface. Consequently, photoactivatable oligonucleotides are powerful tools for investigating nucleic acid-protein interactions involved in numerous biological and pathological processes. In this review, we provide a comprehensive overview of the chemical tools developed so far and the different strategies used for incorporating the most commonly used photoreactive reagents into oligonucleotide probes or nucleic acids. Furthermore, we illustrate their application with several examples including protein binding site mapping, identification of protein binding partners, and in cell studies.

Multivariate Silicification-Assisted Single Enzyme Structure Augmentation for Improved Enzymatic Activity-Stability Trade-Off.

The ability to finely tune/balance the structure and rigidity of enzymes to realize both high enzymatic activity and long-term stability is highly desired but highly challenging. Herein, we propose the concept of the "silicazyme", where solid inorganic silica undergoes controlled hybridization with the fragile enzyme under moderate conditions at the single-enzyme level, thus enabling simultaneous structure augmentation, long-term stability, and high enzymatic activity preservation. A multivariate silicification approach was utilized and occurred around individual enzymes to allow conformal coating. To realize a high activity-stability trade-off the structure flexibility/rigidity of the silicazyme was optimized by a component adjustment ternary (CAT) plot method. Moreover, the multivariate organosilica frameworks bring great advantages, including surface microenvironment adjustability, reversible modification capability, and functional extensibility through the rich chemistry of silica. Overall silicazymes represent a new class of enzymes with promise for catalysis, separations, and nanomedicine.

Catechol-induced covalent modifications modulate the aggregation tendency of α-synuclein: An in-solution and in-silico study.

Parkinson's disease (PD) stands as a challenging neurodegenerative condition characterized by the emergence of Lewy Bodies (LBs), intracellular inclusions within dopaminergic neurons. These LBs harbor various proteins, prominently including α-Synuclein (Syn) aggregates, implicated in disease pathology. A promising avenue in PD treatment involves targeting Syn aggregation. Recent findings from our research have shown that 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET) possess the ability to impede the formation of Syn fibrils by disrupting the aggregation process. Notably, these compounds primarily engage in noncovalent interactions with the protein, leading to the formation of off-pathway oligomers that deter fibril growth. Through proteolysis studies and mass spectrometry (MS) analysis, we have identified potential covalent modifications of Syn in the presence of DOPAC, although the exact site remains elusive. Employing molecular dynamics simulations, we delved into how DOPAC-induced covalent alterations might affect the mechanism of Syn aggregation. Our findings indicate that the addition of a covalent adduct on certain residues enhances fibril flexibility without compromising its secondary structure stability. Furthermore, in the monomeric state, the modified residue fosters novel bonding interactions, thereby influencing long-range interactions between the N- and C-termini of the protein.

Functional metalloenzymes based on myoglobin and neuroglobin that exploit covalent interactions.

Globins, such as myoglobin (Mb) and neuroglobin (Ngb), are ideal protein scaffolds for the design of functional metalloenzymes. To date, numerous approaches have been developed for enzyme design. This review presents a summary of the progress made in the design of functional metalloenzymes based on Mb and Ngb, with a focus on the exploitation of covalent interactions, including coordination bonds and covalent modifications. These include the construction of a metal-binding site, the incorporation of a non-native metal cofactor, the formation of Cys/Tyr-heme covalent links, and the design of disulfide bonds, as well as other Cys-covalent modifications. As exemplified by recent studies from our group and others, the designed metalloenzymes have potential applications in biocatalysis and bioconversions. Furthermore, we discuss the current trends in the design of functional metalloenzymes and highlight the importance of covalent interactions in the design of functional metalloenzymes.

Bifunctional enzyme provides absolute concentration robustness in multisite covalent modification networks.

Biochemical covalent modification networks exhibit a remarkable suite of steady state and dynamical properties such as multistationarity, oscillations, ultrasensitivity and absolute concentration robustness. This paper focuses on conditions required for a network of this type to have a species with absolute concentration robustness. We find that the robustness in a substrate is endowed by its interaction with a bifunctional enzyme, which is an enzyme that has different roles when isolated versus when bound as a substrate-enzyme complex. When isolated, the bifunctional enzyme promotes production of more molecules of the robust species while when bound, the same enzyme facilitates degradation of the robust species. These dual actions produce robustness in the large class of covalent modification networks. For each network of this type, we find the network conditions for the presence of robustness, the species that has robustness, and its robustness value. The unified approach of simultaneously analyzing a large class of networks for a single property, i.e. absolute concentration robustness, reveals the underlying mechanism of the action of bifunctional enzyme while simultaneously providing a precise mathematical description of bifunctionality.

o-Vanillin binds covalently to MAL/TIRAP Lys-210 but independently inhibits TLR2.

Toll-like receptor (TLR) innate immunity signalling protects against pathogens, but excessive or prolonged signalling contributes to a range of inflammatory conditions. Structural information on the TLR cytoplasmic TIR (Toll/interleukin-1 receptor) domains and the downstream adaptor proteins can help us develop inhibitors targeting this pathway. The small molecule o-vanillin has previously been reported as an inhibitor of TLR2 signalling. To study its mechanism of action, we tested its binding to the TIR domain of the TLR adaptor MAL/TIRAP (MALTIR). We show that o-vanillin binds to MALTIR and inhibits its higher-order assembly in vitro. Using NMR approaches, we show that o-vanillin forms a covalent bond with lysine 210 of MAL. We confirm in mouse and human cells that o-vanillin inhibits TLR2 but not TLR4 signalling, independently of MAL, suggesting it may covalently modify TLR2 signalling complexes directly. Reactive aldehyde-containing small molecules such as o-vanillin may target multiple proteins in the cell.

Interaction of soy protein isolate with hydroxytyrosol based on an alkaline method: Implications for structural and functional properties.

This study investigated the effect of different concentrations of hydroxytyrosol (HT) covalently bound to soy protein isolate (SPI) by the alkaline method on the structure and function of the adducts. The amount of polyphenol bound to SPI first increased to a maximum of 42.83 % ± 1.08 % and then decreased. After the covalent binding of HT to SPI, turbidity and in vitro protein digestibility increased and decreased significantly with increasing concentrations of HT added, respectively, and the structure of SPI was changed. The adducts had a maximum solubility of 52.52 % ± 0.33 %, and their water holding capacity reached a maximum of 8.22 ± 0.11 g/g at a concentration of 50 µmol/g of HT. Covalent modification with HT significantly increased the emulsifying and foaming properties and antioxidant activity of SPI; the DPPH and ABTS radical scavenging rates increased by 296.89 % and 33.80 %, respectively, at a concentration of 70 µmol/g of HT.

Interrogating l-fuconate dehydratase with tartronate and 3-hydroxypyruvate reveals subtle differences within the mandelate racemase-subgroup of the enolase superfamily.

Enzymes of the enolase superfamily share a conserved structure and a common partial reaction (i.e., metal-assisted, Brønsted base-catalyzed enol(ate) formation). The architectures of the enolization apparatus at the active sites of the mandelate racemase (MR)-subgroup members MR and l-fuconate dehydratase (FucD) are almost indistinguishable at the structural level. Tartronate and 3-hydroxypyruvate (3-HP) recognize the enolization apparatus and can be used to interrogate the active sites for differences that may not be apparent from structural data. We report a circular dichroism-based assay of FucD activity that monitors the change in ellipticity at 216 nm (Δ[Θ]S-P = 8985 ± 87 deg cm2 mol-1) accompanying the conversion of l-fuconate to 2-keto-3-deoxy-l-fuconate. Tartronate was a linear mixed-type inhibitor of FucD (Ki = 8.4 ± 0.7 mM, αKi = 63 ± 11 mM), binding 18-fold weaker than l-fuconate, compared with 2-fold weaker binding of tartronate by MR relative to mandelate. 3-HP irreversibly inactivated FucD (kinact/KI = 0.018 ± 0.002 M-1s-1) with an efficiency that was ∼4.6 × 103-fold less than that observed with MR. The inactivation arose predominantly from modifications at multiple sites and Tris-HCl, but not l-fuconate, afforded protection against inactivation. Similar to the reaction of 3-HP with MR, 3-HP modified the Brønsted base catalyst (Lys 220) at the active site of FucD, which was facilitated by the Brønsted acid catalyst His 351. Thus, the interactions of tartronate and 3-HP with MR and FucD revealed differences in binding affinity and reactivity that differentiated between the enzymes' enolization apparatuses.

[The Role of Supersulfide in Methylmercury Detoxification].

Methylmercury is a ubiquitous neurotoxic substance present in the environment, and health concerns, especially through the consumption of seafood, remain. Glutathione (GSH)-mediated detoxification and the excretion of methylmercury are known metabolic detoxification pathways. We have also discovered a mechanism by which endogenous super-sulfides convert methylmercury to nontoxic metabolites such as bis-methylmercury sulfide. However, these metabolites are present in very small quantities, and the significance of the detoxification of methylmercury by super-sulfides is not well understood. Methylmercury binds to thiol groups in vivo but can also react with highly reactive selenols (selenocysteine residues). Such covalent bonds (S-mercuration and Se-mercuration) are broken by nucleophilic substitution reactions with other thiol and selenols, however, the contribution of super-sulfides to this substitution reaction is not well understood. Interestingly, a recent study suggested that selenoprotein P, the major selenium transport protein in plasma, binds to methylmercury, however, Se-mercuration was not determined. In this review, we introduce these series of reactions and discuss their involvement with super-sulfides in methylmercury toxicity.

Regulating the Heme Active Site by Covalent Modifications: Two Case Studies of Myoglobin.

Using myoglobin (Mb) as a model protein, we herein developed a facial approach to modifying the heme active site. A cavity was first generated in the heme distal site by F46 C mutation, and the thiol group of Cys46 was then used for covalently linked to exogenous ligands, 1H-1,2,4-triazole-3-thiol and 1-(4-hydroxyphenyl)-1H-pyrrole-2,5-dione. The engineered proteins, termed F46C-triazole Mb and F46C-phenol Mb, respectively, were characterized by X-ray crystallography, spectroscopic and stopped-flow kinetic studies. The results showed that both the heme coordination state and the protein function such as H2 O2 activation and peroxidase activity could be efficiently regulated, which suggests that this approach might be generally applied to the design of functional heme proteins.

Metal-Mediated Ligand Affinity Chemistry (MLAC).

Metal-mediated ligand affinity chemistry (MLAC) enables site-specific protein modification and represents a powerful bioorthogonal strategy. Conventional bioorthogonal methods often involve two steps: (i) incorporation of the bioorthogonal handle (e.g., non-canonical amino acid, enzyme domain, peptide sequences) and (ii) the binding of functional molecules such as drugs, affinity tags, and fluorophores. This two-step protocol often involves genetic manipulation, which makes it impossible to chemically modify endogenous proteins in living systems. Thus, we propose the development of a transition metal-based chemical strategy that is ligand-directed to the endogenous protein of interest in a single step, which we refer to as metal-mediated ligand affinity chemistry (MLAC).

Reversible Postsynthetic Modification in a Metal-Organic Framework.

Postsynthetic modification (PSM) of metal-organic frameworks (MOFs) provides access to functional materials and advanced porous solid engineering. Herein, we report the reversible PSM of a multivariate isoreticular MOF by applying dynamic furan-maleimide Diels-Alder (DA) chemistry. The key step involves incorporating a furan group into the MOF via "click" PSM, which can then undergo repeated cycles of modification and de-modification with maleimides. The structural integrity, crystallinity, and porosity of the furan-appended MOF remained intact even after three consecutive PSM/de-modification cycles using three different functionalized maleimides.

Boosting photocatalytic H2 evolution on UIO-66-NH2/covalent triazine-based frameworks composites by constructing a covalent heterojunction.

Metal-organic frameworks (MOFs) and covalent organic frameworks (COFs) have been proved as efficient catalysts for photocatalytic hydrogen (H2) evolution, thanks to their tunable functionalities, permanent porosity, excellent visible light response, and physicochemical stability. Herein, a series of photocatalysts (termed NUBC) was fabricated by loading different amounts of Zr-UiO-66-NH2 (NU) onto a benzoic acid-modified covalent triazine-based framework (BC) based on post-synthetic covalent modification. The resulting NUBC catalysts exhibited a type-II Z-scheme heterojunction structure formed via the amide covalent bonds between the amine groups on NU and carboxyl groups on BC. The optimal loading of NU on BC is 30 wt.% (30NUBC) and the corresponding photocatalytic H2 evolution rate was 378 µmol h-1 g-1, almost 445 and 2 times than that of NU and BC, respectively. The synergistic effect between the type-II Z-scheme heterojunctions and amide bonds was conducive to boosting visible light harvesting and facilitating charge transportation and separation. Furthermore, the prepared NUBC catalysts show great reusability and stability. Overall, this work sheds light on the design of novel MOF/COF hybrid materials and provides a systematic exploration of their photocatalytic H2 evolution properties.

Epigenetic meets metabolism: novel vulnerabilities to fight cancer.

Histones undergo a plethora of post-translational modifications (PTMs) that regulate nucleosome and chromatin dynamics and thus dictate cell fate. Several evidences suggest that the accumulation of epigenetic alterations is one of the key driving forces triggering aberrant cellular proliferation, invasion, metastasis and chemoresistance pathways. Recently a novel class of histone "non-enzymatic covalent modifications" (NECMs), correlating epigenome landscape and metabolic rewiring, have been described. These modifications are tightly related to cell metabolic fitness and are able to impair chromatin architecture. During metabolic reprogramming, the high metabolic flux induces the accumulation of metabolic intermediate and/or by-products able to react with histone tails altering epigenome homeostasis. The accumulation of histone NECMs is a damaging condition that cancer cells counteracts by overexpressing peculiar "eraser" enzymes capable of removing these modifications preserving histones architecture. In this review we explored the well-established NECMs, emphasizing the role of their corresponding eraser enzymes. Additionally, we provide a parterre of drugs aiming to target those eraser enzymes with the intent to propose novel routes of personalized medicine based on the identification of epi-biomarkers which might be selectively targeted for therapy. Video Abstract.

Reactive architecture profiling with a methyl acyl phosphate electrophile.

Activity-based protein profiling has facilitated the study of the activity of enzymes in proteomes, inhibitor development, and identification of enzymes that share mechanistic and active-site architectural features. Since methyl acyl phosphate monoesters act as electrostatically selective anionic electrophiles for the covalent modification of nucleophiles that reside adjacent to cationic sites in proteins, we synthesized methyl hex-5-ynoyl phosphate (MHP) to broadly target such protein architectures. After treating the soluble proteome of Paucimonas lemoignei with MHP, biotinylating the resulting acylated proteins using click chemistry, enriching the protein adducts using streptavidin, and analyzing the proteins by LC-MS/MS, a set of 240 enzymes and 132 non-enzyme proteins were identified for a wide spectrum of biological processes and from all 7 enzyme classes. Among those enzymes identified, ß-hydroxybutyrate dehydrogenase (PlHBDH) and CTP synthase (E. coli orthologue, EcCTPS) were purified as recombinant enzymes and their rates of inactivation and sites of modification by MHP and methyl acetyl phosphate (MAP) were characterized. MHP reacted more slowly with these proteins than MAP but exhibited greater specificity, despite its lack of multiple binding determinants. Generally, MAP modified more surface residues than MHP. MHP specifically modified Ser 146, Lys 156, and Lys 163 at the active site of PlHBDH. MHP and MAP modified numerous residues of EcCTPS with CTP furnishing the greatest level of protection against MHP- and MAP-dependent modification and inactivation, respectively, followed by ATP and glutamine. Overall, MHP served as an effective probe to identify proteins that are potentially amenable to inhibition by methyl acyl phosphates.

Structural Transitions of Papain-like Cysteine Proteases: Implications for Sensor Development.

The significant role of papain-like cysteine proteases, including papain, cathepsin L and SARS-CoV-2 PLpro, in biomedicine and biotechnology makes them interesting model systems for sensor development. These enzymes have a free thiol group that is suitable for many sensor designs including strong binding to gold nanoparticles or low-molecular-weight inhibitors. Focusing on the importance of the preservation of native protein structure for inhibitor-binding and molecular-imprinting, which has been applied in some efficient examples of sensor development, the aim of this work was to examine the effects of the free-thiol-group's reversible blocking on papain denaturation that is the basis of its activity loss and aggregation. To utilize biophysical methods common in protein structural transitions characterization, such as fluorimetry and high-resolution infrared spectroscopy, low-molecular-weight electrophilic thiol blocking reagent S-Methyl methanethiosulfonate (MMTS) was used in solution. MMTS binding led to a two-fold increase in 8-Anilinonaphthalene-1-sulfonic acid fluorescence, indicating increased hydrophobic residue exposure. A more in-depth analysis showed significant transitions on the secondary structure level upon MMTS binding, mostly characterized by the lowered content of α-helices and unordered structures (either for approximately one third), and the increase in aggregation-specific ß-sheets (from 25 to 52%) in a dose-dependant manner. The recovery of this inhibited protein showed that reversibility of inhibition is accompanied by reversibility of protein denaturation. Nevertheless, a 100-fold molar excess of the inhibitor led to the incomplete recovery of proteolytic activity, which can be explained by irreversible denaturation. The structural stability of the C-terminal ß-sheet rich domain of the papain-like cysteine protease family opens up an interesting possibility to use its foldamers as a strategy for sensor development and other multiple potential applications that rely on the great commercial value of papain-like cysteine proteases.

Comparisons between Myofibrillar Protein-Luteolin Conjugates Fabricated through Covalent and Noncovalent Modification.

Protein-flavonoid conjugation is considered to effectively enhance the functionality of proteins, although how different binding modes affect the conformation and antioxidative properties of these conjugates has yet to be revealed. Herein, myofibrillar protein (MP)-luteolin (Lut) conjugates were noncovalently and covalently constructed using equivalent amounts of Lut (10.00, 20.11, and 69.60 µmol/g protein). Fluorescence quenching confirmed that hydrophobic interactions were the main forces in noncovalent MP-Lut conjugates and that the binding was entropy-driven. Liquid chromatography-tandem mass spectrometry results confirmed that Lut could be covalently grafted with MP after alkaline treatment. Proteomics analysis identified that most graft sites were located on the myosin subunits. Intriguingly, in vitro results showed that the antioxidant activity was barely affected by the MP-Lut binding modes. This work provides a theoretical basis for the application of MP-Lut noncovalent/covalent complexes as functional components.