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In this research article, we report on the strengthening of a non‐classical hydrogen bond (C−H⋅⋅⋅O) by introducing electron withdrawing groups at the carbon atom. The approach is demonstrated on the example of derivatives of the physiological E‐selectin ligand sialyl Lewisx (1, sLex). Its affinity is mainly due to a beneficial entropy term, which is predominantly caused by the pre‐organization of sLex in its binding conformation. We have shown, that among the elements responsible for the pre‐organization, the stabilization by a non‐classical hydrogen bond between the H−C5 of l‐fucose and the ring oxygen O5 of the neighboring d‐galactose moiety is essential and yields 7.4 kJ mol−1. This effect could be further strengthened by replacing l‐fucose by 6,6,6‐trifluoro‐l‐fucose leading to an improved non‐classical H‐bond of 14.9 kJ mol−1, i.e. an improved pre‐organization in the bioactive conformation. For a series of glycomimetics of sLex (1), this outcome could be confirmed by high field NMR‐shifts of the H−C5Fuc, by X‐ray diffraction analysis of glycomimetics co‐crystallized with E‐selectin as well as by isothermal titration calorimetry. Furthermore, the electron‐withdrawing character of the CF3‐group beneficially influences the pharmacokinetic properties of sLex mimetics. Thus, acid‐stability, a prerequisite for gastrointestinal stability, could be substantially improved. [ABSTRACT FROM AUTHOR]

An A1/A2 asymmetric-difunctionalized pillar[5]arene bearing n-bormodecyloxy and propargyloxy groups formed four different supramolecular crystal systems depending on the co-crystallization conditions. A cyclic [c2] daisy chain "double-threaded dimer" was obtained when the macrocycle crystallized from a solution containing chloroform and tolualdehyde. Poly[c2] daisy chain, mediated by the solvent molecules, was formed upon crystallization with N,N-dimethylformamide. The supramolecular self-assemblies in the crystal network of the inclusion complex with adiponitrile guest were strongly dependent on the crystallation solvent. The resulting supramolecular assemblies were fully characterized via single-crystal X-ray diffraction analysis. Results of 1H NMR experiments and isothermal calorimetry titration indicated that their behavior in solution was also solvent-dependent. [ABSTRACT FROM AUTHOR]

BACKGROUND: Dandelion contains hundreds of active compounds capable of inhibiting urease activity, but the individual compounds have not yet been fully identified, and their effects and underlying mechanisms are not clear. The present study aimed to screen the urease inhibition active compounds of dandelion by urease inhibitory activity evaluation HPLC‐tandem mass spectrometry analysis, their mechanism of urease inhibition by polyphenols was explored using enzyme kinetic studies via Lineweaver‐Burk plots. Other investigations included isothermal titration calorimetry and surface plasmon resonance sensing, fluorescence quenching experiments, and single ligand molecular docking and two‐ligand simultaneous docking techniques. RESULTS: The results indicated that the ethyl acetate fraction of dandelion flower exhibited the greatest inhibition (lowest IC50 0.184 ± 0.007 mg mL−1). Chlorogenic acid, caffeic acid and luteolin could be effective urease inhibitors that acted in a non‐competitive inhibition manner. Individually, chlorogenic acid could not only fast bind to urease, but also dissociate rapidly, whereas luteolin might interact with urease with the weakest affinity. The chlorogenic acid‐caffeic acid combination exhibited an additive effect in urease inhibition. However, the chlorogenic acid‐luteolin and caffeic acid‐luteolin combinations exhibited antagonistic effects, with the caffeic acid‐luteolin combination showing greater antagonism. CONCLUSION: The present study reveals that chlorogenic acid, caffeic acid and luteolin are major bioactive compounds for urease inhibition, indicating the molecular mechanisms. The antagonistic effects were observed between luteolin and chlorogenic acid/caffeic acid, and the interactions of the catalytic site and flap may account for the antagonistic effects. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Non‐canonical nucleotides, generated as oxidative metabolic by‐products, significantly threaten the genome integrity of Plasmodium falciparum and thereby, their survival, owing to their mutagenic effects. PfHAM1, an evolutionarily conserved inosine/xanthosine triphosphate pyrophosphohydrolase, maintains nucleotide homeostasis in the malaria parasite by removing non‐canonical nucleotides, although structure–function intricacies are hitherto poorly reported. Here, we report the X‐ray crystal structure of PfHAM1, which revealed a homodimeric structure, additionally validated by size‐exclusion chromatography–multi‐angle light scattering analysis. The two monomeric units in the dimer were aligned in a parallel fashion, and critical residues associated with substrate and metal binding were identified, wherein a notable structural difference was observed in the β‐sheet main frame compared to human inosine triphosphate pyrophosphatase. PfHAM1 exhibited Mg++‐dependent pyrophosphohydrolase activity and the highest binding affinity to dITP compared to other non‐canonical nucleotides as measured by isothermal titration calorimetry. Modifying the pfham1 genomic locus followed by live‐cell imaging of expressed mNeonGreen‐tagged PfHAM1 demonstrated its ubiquitous presence in the cytoplasm across erythrocytic stages with greater expression in trophozoites and schizonts. Interestingly, CRISPR‐Cas9/DiCre recombinase‐guided pfham1‐null P. falciparum survived in culture under standard growth conditions, indicating its assistive role in non‐canonical nucleotide clearance during intra‐erythrocytic stages. This is the first comprehensive structural and functional report of PfHAM1, an atypical nucleotide‐cleansing enzyme in P. falciparum. [ABSTRACT FROM AUTHOR]

Multivalency is a fundamental principle in nature that leads to high‐affinity intermolecular recognition through multiple cooperative interactions that overcome the weak binding of individual constituents. For example, multivalency plays a critical role in lectin‐carbohydrate interactions that participate in many essential biological processes. Designing high‐affinity multivalent glycoconjugates that engage lectins results in systems with the potential to disrupt these biological processes, offering promising applications in therapeutic design and bioengineering. Here, a versatile and tunable synthetic platform for the synthesis of metallosupramolecular glycoassemblies is presented that leverages subcomponent self‐assembly, which employs metal ion templates to generate complex supramolecular architectures from simple precursors in one pot. Through ligand design, this approach provides precise control over molecular parameters such as size, shape, flexibility, valency, and charge, which afforded a diverse family of well‐defined hybrid glyconanoassemblies. Evaluation of these complexes as multivalent binders to Concanavalin A (Con A) by isothermal titration calorimetry (ITC) demonstrates the optimal saccharide tether length and the effect of electrostatics on protein affinity, revealing insights into the impact of synthetic design on molecular recognition. The presented studies offer an enhanced understanding of structure‐function relationships governing lectin‐saccharide interactions at the molecular level and guide a systematic approach towards optimizing glyconanoassembly binding parameters. [ABSTRACT FROM AUTHOR]

[Display omitted] • MO-2097 is a novel synthetic chiral-free HIF-1α inhibitor targeting hnRNPA2B1 which is elevated in cancer patients. • MO-2097 showed significant anticancer results on patient-derived cancer organoids. • MO-2097 is a novel chemotype and a promising potential anticancer therapeutic. Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator mediating adaptive responses to hypoxia. It is up-regulated in the tumor microenvironment and recognized as an effective anticancer drug target. Previously, we discovered that the natural compound moracin-O and its synthetic derivative MO-460 inhibited HIF-1α via hnRNPA2B1. This study aimed to develop novel HIF-1 inhibitors for cancer chemotherapy by harnessing the potential of the natural products moracins-O and P. In an ongoing search for novel HIF-1 inhibitors, a series of nature-inspired benzofurans with modifications on the chiral rings of moracins-O and P were synthesized. They showed improved chemical tractability and were evaluated for their inhibitory activity on HIF-1α accumulation under hypoxic conditions in HeLa CCL2 cells. The most potent derivative's chemical-based toxicities, binding affinities, and in vivo anti-tumorigenic effects were evaluated. Further, we examined whether our compound, MO-2097, exhibited anticancer effects in three-dimensional cultured organoids. Herein, we identified a novel synthetic chiral-free compound, MO-2097, with reduced structural complexity and increased efficiency. MO-2097 exhibited inhibitory effects on hypoxia-induced HIF-1α accumulation in HeLa CCL2 cells via inhibition of hnRNPA2B1 protein, whose binding affinities were confirmed by isothermal titration calorimetry analysis. In addition, MO-2097 demonstrated in vivo efficacy and biocompatibility in a BALB/c mice xenograft model. The immunohistochemistry staining of MO-2097-treated tissues showed decreased expression of HIF-1α and increased levels of apoptosis marker cleaved caspase 3, confirming in vivo efficacy. Furthermore, we confirmed that MO-2097 works effectively in cancer patient-based organoid models. MO-2097 represents a promising new generation of chemotherapeutic agents targeting HIF-1α inhibition via hnRNPA2B1, requiring further investigation. [ABSTRACT FROM AUTHOR]

The paper presents a physicochemical study of the water-soluble adduct of C60 with l-arginine, including a quantum chemical determination of the stability of isomers, measurement of antiradical activity towards the ABTS radical, determination of the lipophilicity coefficient, particle size distribution in aqueous solutions and calculation of ζ-potentials. A separate part of the article is devoted to the study of the effect of the C60 adduct with l-arginine on DNA in in vitro experiments (determination of the binding constant using isothermal titration calorimetry, study of genotoxicity, DNA melting temperature, effect on DNA circular dichroism spectra). Data are also presented on the biodistribution of the C60 adduct with l-arginine in the organs and tissues of animals and its effect on clinical and biochemical blood parameters. The mutagenic effect of the C60 adduct with l-arginine was determined in vivo on a model of mice abnormal sperm heads. It was shown that the adduct is in a neutral state in physiological solution, has an affinity to both aqueous and lipid phases (logPow = 0.23), and forms a complex with DNA (Ka = 4.59 106 M −1), while it does not have cyto- and genotoxicity in vitro and mutagenicity in vivo. [ABSTRACT FROM AUTHOR]

Mutations in parkin and PINK1 cause early-onset Parkinson's disease (EOPD). The ubiquitin ligase parkin is recruited to damaged mitochondria and activated by PINK1, a kinase that phosphorylates ubiquitin and the ubiquitin-like domain of parkin. Activated phospho-parkin then ubiquitinates mitochondrial proteins to target the damaged organelle for degradation. Here, we present the mechanism of activation of a new class of small molecule allosteric modulators that enhance parkin activity. The compounds act as molecular glues to enhance the ability of phospho-ubiquitin (pUb) to activate parkin. Ubiquitination assays and isothermal titration calorimetry with the most active compound (BIO-2007817) identify the mechanism of action. We present the crystal structure of a closely related compound (BIO-1975900) bound to a complex of parkin and two pUb molecules. The compound binds next to pUb on RING0 and contacts both proteins. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments confirm that activation occurs through release of the catalytic Rcat domain. In organello and mitophagy assays demonstrate that BIO-2007817 partially rescues the activity of parkin EOPD mutants, R42P and V56E, offering a basis for the design of activators as therapeutics for Parkinson's disease. Parkin is a ubiquitin ligase that protects against early-onset Parkinson's disease. Here, the authors show a molecular glue that promotes binding of phosphorylated ubiquitin to parkin and rescues the mitophagy defect of mutations in the parkin ubiquitin-like domain. [ABSTRACT FROM AUTHOR]

The significance and necessity of separating enantiomers in food, pharmaceuticals, pesticides, and other samples remains constant and unrelenting. The successful chiral separation usually includes the application of a chiral auxiliary compound, known also as a chiral selector (CS), that forms complexes with enantiomers of different physicochemical properties, enabling efficient separation. While both native and substituted cyclodextrins (CDs) are commonly used as CSs, β-CD is undoubtedly the most popular one among them. This review includes recent advancements in the application of β-CD as a CS. While the theoretical background behind the enantioseparation is also part of this work, the main emphasis is put on the factors that affect the efficacy of this process such as temperature, pH, solvent, and the choice of other additives. Also, the different analytical methods: Nuclear Magnetic Resonance (NMR) spectroscopy, Capillary Electrophoresis (CE), fluorescence spectroscopy (FS), High-Performance Liquid Chromatography (HPLC), Isothermal Titration Calorimetry (ITC), and UV–vis spectroscopy, used for enantioseparation with the aid of β-CD as CS, are thoroughly compared. Also, since some of the chiral compounds have been studied in the context of their enantioseparation more than once, those works are compared and critically analyzed. In conclusion, while β-CD can be in most cases used as CS, the choice of the experimental conditions and method of analysis is crucial to achieve the success. [ABSTRACT FROM AUTHOR]

In the realm of disease vectors and agricultural pest management, insecticides play a crucial role in preserving global health and ensuring food security. The pervasive use, particularly of organophosphates (OPs), has given rise to a substantial challenge in the form of insecticide resistance. Carboxylesterases emerge as key contributors to OP resistance, owing to their ability to sequester or hydrolyze these chemicals. Consequently, carboxylesterase enzymes become attractive targets for the development of novel insecticides. Inhibiting these enzymes holds the potential to restore the efficacy of OPs against which resistance has developed. This study aimed to screen the FooDB library to identify potent inhibitory compounds targeting carboxylesterase, Ha006a from the agricultural pest Helicoverpa armigera. The ultimate objective is to develop effective interventions for pest control. The compounds with the highest scores underwent evaluation through docking studies and pharmacophore analysis. Among them, four phytochemicals—donepezil, protopine, 3′,4′,5,7-tetramethoxyflavone, and piperine—demonstrated favorable binding affinity. The Ha006a-ligand complexes were subsequently validated through molecular dynamics simulations. Biochemical analysis, encompassing determination of IC50 values, complemented by analysis of thermostability through Differential Scanning Calorimetry and interaction kinetics through Isothermal Titration Calorimetry was conducted. This study comprehensively characterizes Ha006a-ligand complexes through bioinformatics, biochemical, and biophysical methods. This investigation highlights 3′,4′,5,7-tetramethoxyflavone as the most effective inhibitor, suggesting its potential for synergistic testing with OPs. Consequently, these inhibitors offer a promising solution to OP resistance and address environmental concerns associated with excessive insecticide usage, enabling a significant reduction in their overuse. [ABSTRACT FROM AUTHOR]

AbstractPolyphenolic compounds have received much attention due to their major function as antioxidants. Sinapic acid (SA) is an orally bioavailable phytochemical with excellent free radical scavenging property transported in the blood by human serum albumin (HSA) which is the chief carrier protein found abundantly in human plasma. SA contains a single phenolic group. This study investigates the binding and interaction mechanism between SA and HSA by multiple spectroscopic and calorimetric techniques, such as UV/visible absorption spectroscopy, intrinsic fluorescence studies, Fourier transform infrared (FTIR) spectroscopy and isothermal titration calorimetry (ITC). UV/visible absorption spectroscopy showed hyperchromicity in the absorption spectra, suggestive of structural change due to complex formation between HSA and SA. Intrinsic fluorescence measurements, performed at three different temperatures, showed quenching of the fluorescence spectra and the mechanism of quenching was static in nature which occurred due to ground state complex formation. The FTIR results demonstrated a 16% decrease in α-helical content of the secondary structure of HSA in the presence of SA. Hydrogen bonds and hydrophobic forces were the main forces of interaction, according to the thermodynamic characteristics found through ITC. Moreover, the data obtained through ITC indicated strong binding affinity between HSA and SA and supported the exothermic and spontaneous nature of the interaction. [ABSTRACT FROM AUTHOR]

Ferrous iron (Fe2+) is required for the growth and virulence of many pathogenic bacteria, including Vibrio cholerae (Vc), the causative agent of the disease cholera. For this bacterium, Feo is the primary system that transports Fe2+ into the cytosol. FeoB, the main component of this system, is regulated by a soluble cytosolic domain termed NFeoB. Recent reanalysis has shown that NFeoBs can be classified as either GTP-specific or NTP-promiscuous, but the structural and mechanistic bases for these differences were not known. To explore this intriguing property of FeoB, we solved the X-ray crystal structures of VcNFeoB in both the apo and the GDP-bound forms. Surprisingly, this promiscuous NTPase displayed a canonical NFeoB G-protein fold like GTP-specific NFeoBs. Using structural bioinformatics, we hypothesized that residues surrounding the nucleobase could be important for both nucleotide affinity and specificity. We then solved the X-ray crystal structures of N150T VcNFeoB in the apo and GDP-bound forms to reveal H-bonding differences surrounding the guanine nucleobase. Interestingly, isothermal titration calorimetry revealed similar binding thermodynamics of the WT and N150T proteins to guanine nucleotides, while the behavior in the presence of adenine nucleotides was dramatically different. AlphaFold models of VcNFeoB in the presence of ADP and ATP showed important conformational changes that contribute to nucleotide specificity among FeoBs. Combined, these results provide a structural framework for understanding FeoB nucleotide promiscuity, which could be an adaptive measure utilized by pathogens to ensure adequate levels of intracellular iron across multiple metabolic landscapes. [ABSTRACT FROM AUTHOR]

NEIL1 is a DNA glycosylase that recognizes and initiates base excision repair of oxidized bases. The ubiquitous ssDNA binding scaffolding protein, replication protein A (RPA), modulates NEIL1 activity in a manner that depends on DNA structure. Interaction between NEIL1 and RPA has been reported, but the molecular basis of this interaction has yet to be investigated. Using a combination of NMR spectroscopy and isothermal titration calorimetry (ITC), we show that NEIL1 interacts with RPA through two contact points. An interaction with the RPA32C protein recruitment domain was mapped to a motif in the common interaction domain (CID) of NEIL1 and a dissociation constant (Kd) of 200 nM was measured. A substantially weaker secondary interaction with the tandem RPA70AB ssDNA binding domains was also mapped to the CID. Together these two contact points reveal NEIL1 has a high overall affinity (Kd ( 20 nM) for RPA. A homology model of the complex of RPA32C with the NEIL1 RPA binding motif in the CID was generated and used to design a set of mutations in NEIL1 to disrupt the interaction, which was confirmed by ITC. The mutant NEIL1 remains catalytically active against a thymine glycol lesion in duplex DNA in vitro. Testing the functional effect of disrupting the NEIL1-RPA interaction in vivo using a Fluorescence Multiplex-Host Cell Reactivation (FM-HCR) reporter assay revealed an unexpected role for NEIL1 in nucleotide excision repair. These findings are discussed in the context of the role of NEIL1 in replicationassociated repair. [ABSTRACT FROM AUTHOR]

Abstract: Cell surface signaling (CSS) is a means of rapidly adjusting transcription in response to extracellular stimuli in Gram-negative bacteria. The pseudobactin BN7/8 uptake (Pup) system not only imports iron but also upregulates its own transcription through CSS in Pseudomonas capeferrum. In the absence of ferric pseudobactin BN7/8, the signaling components are maintained in a resting state via the formation of a periplasmic complex between the N-terminal signaling domain (NTSD) of the outer membrane iron-transporter, PupB, and the C-terminal CSS domain (CCSSD) of the sigma regulator, PupR. The previously determined 1.6 Å crystal structure of this periplasmic complex has allowed us to probe the structural and thermodynamic consequences of mutating key interfacial residues. In this report, we describe the solution structure of the PupB NTSD and use Nuclear Magnetic Resonance spectroscopy, Isothermal Titration Calorimetry, and Circular Dichroism spectroscopy together with thermal denaturation to investigate whether three PupB point mutations, Q69K, H72D, and L74A, influence the interaction merely due to the chemical nature of the amino acid substitution or also cause changes in overall protein structure. Our results demonstrate that binding to the PupR CCSSD does not alter the structure of PupB NTSD and that the individual mutations have only minor effects on structure. The mutations generally lower thermodynamic stability of the NTSD and weaken binding to the CCSSD. These findings validate the X-ray crystal structure interface, emphasizing the importance of amino acid chemical nature at the interface. [ABSTRACT FROM AUTHOR]

Nanocomplexes, which possess immense potential to function as nanovehicles, can link diverse ligand compounds. The objective of the present study was to design and characterize resveratrol (RSV)- and tocopherol (TOC)-loaded 11S quinoa seed protein nanocomplexes. Firstly, molecular docking was performed to describe the probable binding sites between protein and ligands, and binding energies of −5.6 and −6.2 kcal/mol were found for RSV and TOC, respectively. Isothermal titration calorimetry allowed us to obtain the thermodynamic parameters that described the molecular interactions between RSV or TOC with the protein, finding the complexation process to be exothermic and spontaneous. 11S globulin intrinsic fluorescence spectra showed quenching effects exerted by RSV and TOC, demonstrating protein–bioactive compound interactions. The application of Stern–Volmer, Scatchard, and Förster resonance energy transfer models confirmed static quenching and allowed us to obtain parameters that described the 11S-RSV and 11S-TOC complexation processes. RSV has a higher tendency to bind 11S globulin according to ITC and fluorescence analysis. Secondly, the protein aggregation induced by bioactive compound interactions was confirmed by dynamic light scattering and atomic force microscopy, with diameters <150 nm detected by both techniques. Finally, it was found that the antioxidant capacity of a single 11S globulin did not decrease; meanwhile, it was additive for 11S-RSV. These nanocomplexes could constitute a real platform for the design of nutraceutical products. [ABSTRACT FROM AUTHOR]

A series of biotin-functionalized transition metal complexes was prepared by iClick reaction from the corresponding azido complexes with a novel alkyne-functionalized biotin derivative ([Au(triazolatoR,R′)(PPh3)], [Pt(dpb)(triazolatoR,R′)], [Pt(triazolatoR,R′)(terpy)]PF6, and [Ir(ppy)(triazolatoR,R′)(terpy)]PF6 with dpb = 1,3-di(2-pyridyl)benzene, ppy = 2-phenylpyridine, and terpy = 2,2′:6′,2′′-terpyridine and R = C6H5, R′ = biotin). The complexes were compared to reference compounds lacking the biotin moiety. The binding affinity toward avidin and streptavidin was evaluated with the HABA assay as well as isothermal titration calorimetry (ITC). All compounds exhibit the same binding stoichiometry of complex-to-avidin of 4:1, but the ITC results show that the octahedral Ir(III) compound exhibits a higher binding affinity than the square-planar Pt(II) complex. The antibacterial activity of the compounds was evaluated on a series of Gram-negative and Gram-positive bacterial strains. In particular, the neutral Au(I) and Pt(II) complexes showed significant antibacterial activity against Staphylococcus aureus and Enterococcus faecium at very low micromolar concentrations. The cytotoxicity against a range of eukaryotic cell lines was studied and revealed that the octahedral Ir(III) complex was non-toxic, while the square-planar Pt(II) and linear Au(I) complexes displayed non-selective micromolar activity. [ABSTRACT FROM AUTHOR]

The limited availability of efficient treatments for Candida infections and the increased emergence of antifungal-resistant strains stimulates the search for new antifungal agents. We have previously isolated a sunflower mannose-binding lectin (Helja) with antifungal activity against Candida albicans , capable of binding mannose-bearing oligosaccharides exposed on the cell surface. This work aimed to investigate the biological and biophysical basis of Helja's binding to C. albicans cell wall mannans and its influence on the fungicidal activity of the lectin. We evaluated the interaction of Helja with the cell wall mannans extracted from the isogenic parental strain (WT) and a glycosylation-defective C. albicans with altered cell wall phosphomannosylation (mnn4 ∆ null mutants) and investigated its antifungal effect. Helja exhibited stronger antifungal activity on the mutant strain, showing greater inhibition of fungal growth, loss of cell viability, morphological alteration, and formation of clusters with agglutinated cells. This differential biological activity of Helja was correlated with the biophysical parameters determined by solid phase assays and isothermal titration calorimetry, which demonstrated that the lectin established stronger interactions with the cell wall mannans of the mnn4 ∆ null mutant than with the WT strain. In conclusion, our results provide new evidence on the nature of the Helja molecular interactions with cell wall components, i.e. phosphomannan, and its impact on the antifungal activity. This study highlights the relevance of plant lectins in the design of effective antifungal therapies. [ABSTRACT FROM AUTHOR]

The Atg8-family proteins (MAP1LC3/LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2) play a pivotal role in macroautophagy/autophagy through their ability to help form autophagosomes. Although autophagosomes form in the cytoplasm, nuclear levels of the Atg8-family proteins are significant. Recently, the nuclear/cytoplasmic shuttling of LC3B was shown to require deacetylation of two Lys residues (K49 and K51 in LC3B), which are conserved in Atg8-family proteins. To exit the nucleus, deacetylated LC3B must bind TP53INP2/DOR (tumor protein p53 inducible nuclear protein 2) through interaction with the LC3-interacting region (LIR) of TP53INP2 (TP53INP2LIR). To examine their selectivity for TP53INP2 and the role of the conserved Lys residues in Atg8-family proteins, we prepared the six human Atg8-family proteins and acetylated variants of LC3A and GABARAP for biophysical and structural characterization of their interactions with the TP53INP2LIR. Isothermal titration calorimetry (ITC) experiments demonstrate that this LIR binds preferentially to GABARAP subfamily proteins, and that only acetylation of the second Lys residue reduces binding to GABARAP and LC3A. Crystal structures of complexes with GABARAP and LC3A (acetylated and deacetylated) define a β-sheet in the TP53INP2LIR that determines the GABARAP selectivity and establishes the importance of acetylation at the second Lys. The in vitro results were confirmed in cells using acetyl-mimetic variants of GABARAP and LC3A to examine nuclear/cytoplasmic shuttling and colocalization with TP53INP2. Together, the results demonstrate that TP53INP2 shows selectivity to the GABARAP subfamily and acetylation at the second Lys of GABARAP and LC3A disrupts key interactions with TP53INP2 required for their nuclear/cytoplasmic shuttling. [ABSTRACT FROM AUTHOR]

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in Brassica vegetables, which can induce the host plant to produce salicylic acid and 4-hydroxybenzoic acid (4-HBA) during infection. Xcc was previously shown to sense and degrade host plant-derived 4-HBA via the sensor PobR and a PobA-dependent pathway. The degradation of 4-HBA is associated with Xcc virulence in cabbage. The present study generated a reporter strain XC1::PpobA-gusA to monitor pobA transcription. 4-HBA-like compounds were screened for their ability to interfere with pobA transcription. Benzoic acid (BA) was found to efficiently decrease pobA transcription in a dose-dependent manner. Xcc neither produced nor degraded BA; however, the exogenous addition of BA to the 4-HBA-containing Xcc culture significantly decreased the 4-HBA degradation rate. Furthermore, addition of BA into the Xcc culture did not significantly affect the transcription of pobA or pobR; however, addition of BA into the 4-HBA-containing culture significantly decreased the transcription of both genes. Isothermal titration calorimetry and an electrophoretic mobility shift assay revealed that BA binds to PobR with a moderate affinity, which interfered with the binding of 4-HBA/PobR complex to the pobA promoter and thereby inhibiting pobA transcription and 4-HBA degradation. The endogenous BA level of the infected cabbage leaves increased in response to Xcc infection. In the presence of BA, the virulence of Xcc on cabbage decreased significantly. Taken together, these results suggest that cabbage utilizes BA to interfere with 4-HBA degradation, thereby reducing Xcc virulence. Thus, BA has the potential to be developed as a bactericide against Xcc infection. [ABSTRACT FROM AUTHOR]

Malaria, an ancient mosquito-borne illness caused by Plasmodium parasites, is mostly treated with Artemisinin Combination Therapy (ACT). However, Single Nucleotide Polymorphisms (SNPs) mutations in the P. falciparum Kelch 13 (PfK13) protein have been associated with artemisinin resistance (ART-R). Therefore, this study aims to generate PfK13 recombinant proteins incorporating of two specific SNPs mutations, PfK13-V494I and PfK13-N537I, and subsequently analyze their binding interactions with artemisinin (ART). The recombinant proteins of PfK13 mutations and the Wild Type (WT) variant were expressed utilizing a standard protein expression protocol with modifications and subsequently purified via IMAC and confirmed with SDS-PAGE analysis and Orbitrap tandem mass spectrometry. The binding interactions between PfK13-V494I and PfK13-N537I propeller domain proteins ART were assessed through Isothermal Titration Calorimetry (ITC) and subsequently validated using fluorescence spectrometry. The protein concentrations obtained were 0.3 mg/ml for PfK13-WT, 0.18 mg/ml for PfK13-V494I, and 0.28 mg/ml for PfK13-N537I. Results obtained for binding interaction revealed an increased fluorescence intensity in the mutants PfK13-N537I (83 a.u.) and PfK13-V494I (143 a.u.) compared to PfK13-WT (33 a.u.), indicating increased exposure of surface proteins because of the looser binding between PfK13 protein mutants with ART. This shows that the PfK13 mutations may induce alterations in the binding interaction with ART, potentially leading to reduced effectiveness of ART and ultimately contributing to ART-R. However, this study only elucidated one facet of the contributing factors that could serve as potential indicators for ART-R and further investigation should be pursued in the future to comprehensively explore this complex mechanism of ART-R. [ABSTRACT FROM AUTHOR]

The 2011 discovery of the first rare earth-dependent enzyme in methylotrophic Methylobacterium extorquens AM1 prompted intensive research toward understanding the unique chemistry at play in these systems. This enzyme, an alcohol dehydrogenase (ADH), features a La3+ ion closely associated with redox-active coenzyme pyrroloquinoline quinone (PQQ) and is structurally homologous to the Ca2+-dependent ADH from the same organism. AM1 also produces a periplasmic PQQ-binding protein, PqqT, which we have now structurally characterized to 1.46-Å resolution by X-ray diffraction. This crystal structure reveals a Lys residue hydrogen-bonded to PQQ at the site analogously occupied by a Lewis acidic cation in ADH. Accordingly, we prepared K142A-and K142D-PqqT variants to assess the relevance of this site toward metal binding. Isothermal titration calorimetry experiments and titrations monitored by UV-Vis absorption and emission spectroscopies support that K142D-PqqT binds tightly (Kd = 0.6 ± 0.2 μM) to La3+ in the presence of bound PQQ and produces spectral signatures consistent with those of ADH enzymes. These spectral signatures are not observed for WT-or K142A-variants or upon addition of Ca2+ to PQQ ⊏ K142D-PqqT. Addition of benzyl alcohol to La3+-bound PQQ ⊏ K142D-PqqT (but not Ca2+-bound PQQ ⊏ K142D-PqqT, or La3+-bound PQQ ⊏ WT-PqqT) produces spectroscopic changes associated with PQQ reduction, and chemical trapping experiments reveal the production of benzaldehyde, supporting ADH activity. By creating a metal binding site that mimics native ADH enzymes, we present a rare earth-dependent artificial metalloenzyme primed for future mechanistic, biocatalytic, and biosensing applications. [ABSTRACT FROM AUTHOR]

TNIP1 has been increasingly recognized as a security check to finely adjust the rate of mitophagy by disrupting the recycling of the Unc-51–like kinase complex during autophagosome formation. Through tank-binding kinase 1–mediated phosphorylation of the TNIP1 FIP200 interacting region (FIR) motif, the binding affinity of TNIP1 for FIP200, a component of the Unc-51–like kinase complex, is enhanced, allowing TNIP1 to outcompete autophagy receptors. Consequently, FIP200 is released from the autophagosome, facilitating further autophagosome expansion. However, the molecular basis by which FIP200 utilizes its claw domain to distinguish the phosphorylation status of residues in the TNIP1 FIR motif for recognition is not well understood. Here, we elucidated multiple crystal structures of the complex formed by the FIP200 claw domain and various phosphorylated TNIP1 FIR peptides. Structural and isothermal titration calorimetry analyses identified the crucial residues in the FIP200 claw domain responsible for the specific recognition of phosphorylated TNIP1 FIR peptides. Additionally, utilizing structural comparison and molecular dynamics simulation data, we demonstrated that the C-terminal tail of TNIP1 peptide affected its binding to the FIP200 claw domain. Moreover, the phosphorylation of TNIP1 Ser123 enabled the peptide to effectively compete with the peptide p-CCPG1 (the FIR motif of the autophagy receptor CCPG1) for binding with the FIP200 claw domain. Overall, our work provides a comprehensive understanding of the specific recognition of phosphorylated TNIP1 by the FIP200 claw domain, marking an initial step toward fully understanding the molecular mechanism underlying the TNIP1-dependent inhibition of mitophagy. [ABSTRACT FROM AUTHOR]

The dimeric architecture of tandem-repeat type galectins, such as galectin-4 (Gal-4), modulates their biological activities, although the underlying molecular mechanisms have remained elusive. Emerging evidence show that tandem-repeat galectins play an important role in innate immunity by recognizing carbohydrate antigens present on the surface of certain pathogens, which very often mimic the structures of the human self-glycan antigens. Herein, we have analyzed the binding preferences of the C-domain of Gal-4 (Gal-4C) toward the ABH-carbohydrate histo-blood antigens with different core presentations and their recognition features have been rationalized by using a combined experimental approach including NMR, solid-phase and hemagglutination assays, and molecular modeling. The data show that Gal-4C prefers A over B antigens (two-fold in affinity), contrary to the N-domain (Gal-4N), although both domains share the same preference for the type-6 presentations. The behavior of the full-length Gal-4 (Gal-4FL) tandem-repeat form has been additionally scrutinized. Isothermal titration calorimetry and NMR data demonstrate that both domains within full-length Gal-4 bind to the histo-blood antigens independently of each other, with no communication between them. In this context, the heterodimeric architecture does not play any major role, apart from the complementary A and B antigen binding preferences. However, upon binding to a bacterial lipopolysaccharide containing a multivalent version of an Hantigen mimetic as O-antigen, the significance of the galectin architecture was revealed. Indeed, our data point to the linker peptide domain and the F-face of the C-domain as key elements that provide Gal-4 with the ability to cross-link multivalent ligands, beyond the glycan binding capacity of the dimer. [ABSTRACT FROM AUTHOR]

Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg2+ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg2+. Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg2+, while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg2+ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg2+. Conclusively, the presence of Mg2+ induces a conformation in KpPrAT that favours nucleotide binding. [ABSTRACT FROM AUTHOR]

Anthelmintic resistance in gastrointestinal nematodes produces substantial challenges to agriculture, and new strategies for nematode control in livestock animals are called for. Natural compounds, including tannins, with proven anthelmintic activity could be a functional option as structurally diverse complementary compounds to be used alongside commercial anthelmintics. However, the dual use of two anthelmintic components requires an understanding of the pharmacological effects of the combination, while information concerning the interactions between plant-based polyphenols and commercial anthelmintics is scarce. We studied the direct interactions of proanthocyanidins (PAs, syn. condensed tannins) and a commercial anthelmintic thiabendazole, as a model substance of benzimidazoles, by isothermal titration calorimetry (ITC). Our results show evidence of a direct interaction of an exothermic nature with observed enthalpy changes ranging from 0 to −30 kJ/mol. The strength of the interaction between PAs and thiabendazole is mediated by structural characteristics of the PAs with the strongest positive correlation originating from the presence of galloyl groups and the increased degree of polymerization. [ABSTRACT FROM AUTHOR]

Introduction: The design of carbohydrate-binding agents (artificial carbohydrate receptors) that enable selective and effective biomimetic recognition via noncovalent interactions is aimed either at a better understanding of natural recognition phenomena or at various potential applications in medicine and other fields. Although very interesting artificial receptors have been developed, the exact prediction of the receptor selectivity remains a challenge. Results and Methods: A molecular architecture based on a 1,3,5-substituted 2,4,6-triethylbenzene backbone bearing two aminopyridine- or aminopyrimidine-based recognition units and a purine moiety, which acts as both a hydrogen bonding site and a bridging component for the incorporation of additional substituents, has proved to be very useful for the development of effective carbohydrate-binding agents. This type of compounds has the ability to bind suitable carbohydrates through combined noncovalent interactions, where CH···π interactions can be formed on both faces of the carbohydrate substrate. The successful syntheses of the target compounds can be realized by the use of microwave irradiation and sealed tubes. The performed binding studies included ¹ H NMR spectroscopic titrations and measurements by isothermal titration calorimetry. Conclusion: The new compounds were developed as artificial receptors especially for carbohydrates with an all-equatorial substitution pattern and have the ability to predictably form strong 1:1 complexes with a suitable substrate. The use of the purine moiety in the construction of carbohydrate receptors with a 1,3,5-substituted 2,4,6-triethylbenzene backbone has proved to be a very promising approach. The possibilities for structural variation of this molecular architecture are manifold. As a result, a wide range of compounds can be synthesized to perform extensive studies on the relationships between structure and binding efficiency [ABSTRACT FROM AUTHOR]

Sinorhizobium meliloti senses nutrients and compounds exuded from alfalfa host roots and coordinates an excitation, termination, and adaptation pathway during chemotaxis. We investigated the role of the novel S. meliloti chemotaxis protein CheT. While CheT and the Escherichia coli phosphatase CheZ share little sequence homology, CheT is predicted to possess an α‐helix with a DXXXQ phosphatase motif. Phosphorylation assays demonstrated that CheT dephosphorylates the phosphate‐sink response regulator, CheY1~P by enhancing its decay two‐fold but does not affect the motor response regulator CheY2~P. Isothermal Titration Calorimetry (ITC) experiments revealed that CheT binds to a phosphomimic of CheY1~P with a KD of 2.9 μM, which is 25‐fold stronger than its binding to CheY1. Dissimilar chemotaxis phenotypes of the ΔcheT mutant and cheT DXXXQ phosphatase mutants led to the hypothesis that CheT exerts additional function(s). A screen for potential binding partners of CheT revealed that it forms a complex with the methyltransferase CheR. ITC experiments confirmed CheT/CheR binding with a KD of 19 μM, and a SEC‐MALS analysis determined a 1:1 and 2:1 CheT/CheR complex formation. Although they did not affect each other's enzymatic activity, CheT binding to CheY1~P and CheR may serve as a link between signal termination and sensory adaptation. [ABSTRACT FROM AUTHOR]

This study investigates unexpected competitive host–guest interactions of β‐cyclodextrin (β‐CD), which can occur with polymers in aqueous solution, using the examples of the two polymers poly(oligo(ethylene glycol) methyl ether methacrylate) and poly(glycerol mono methacrylate). Systematic structural modifications of the polymer provide insight into the host–guest interaction with β‐CD and the competition between side chains and end groups such as hydrophobic end groups remaining from reversible addition fragmentation chain transfer polymerization or intentionally implemented molecular recognition units such as arylazopyrazole photoswitches. [ABSTRACT FROM AUTHOR]

Copyright of Science & Technology of Food Industry is the property of Science & Technology of Food Industry Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Soybean (Glycine max) and mung bean (Vigna radiata) are key legumes with global importance, but their mechanisms for coping with cold stress—a major challenge in agriculture—have not been thoroughly investigated, especially in a comparative study. This research aimed to fill this gap by examining how these two major legumes respond differently to cold stress and exploring the role of uniconazole, a potential stress mitigator. Our comprehensive approach involved transcriptomic and metabolomic analyses, revealing distinct responses between soybean and mung bean under cold stress conditions. Notably, uniconazole was found to significantly enhance cold tolerance in mung bean by upregulating genes associated with photosynthesis, while its impact on soybean was either negligible or adverse. To further understand the molecular interactions, we utilized advanced machine learning algorithms for protein structure prediction, focusing on photosynthetic pathways. This enabled us to identify LOC106780309 as a direct binding target for uniconazole, confirmed through isothermal titration calorimetry. This research establishes a new comparative approach to explore how soybean and mung bean adapt to cold stress, offers key insights to improve the hardiness of legumes against environmental challenges, and contributes to sustainable agricultural practices and food security. [ABSTRACT FROM AUTHOR]

A quantitative understanding of thermodynamic effects of avidity in biomolecular interactions is important. Herein, we synthesized discrete glycooligomers and evaluated their interactions with a model protein using isothermal titration calorimetry. The dimeric glycooligomer exhibited higher binding constants compared to the glycomonomer, attributed to the reduced conformational entropy loss through local presentation of multiple carbohydrate units. Conversely, divalent glycoligands with polyethylene glycol linkers, aiming for multivalent binding, showed enhanced interactions through increased enthalpy. These findings emphasize the importance of distinguishing between the "local avidity" and the "multipoint avidity". [ABSTRACT FROM AUTHOR]

Conformationally flexible tertiary amine – thiourea−urea catalysts 1 and 2 for the Mannich reaction between imines and malonate esters are efficiently inhibited by quaternary ammonium halides. NMR titrations, isothermal titration calorimetry (ITC) and NOE experiments showed that the catalysts bind chloride and bromide ions with relatively high affinities (K=103–105 M−1 in acetonitrile). The halide ions not only block the active site of the catalysts, but they also induce refolding into catalytically inactive conformations upon complexation in an allosteric‐like event. At substoichiometric inhibitor:catalyst ratios, the catalysts displayed hypersensitivity to the inhibitors, with overall rates that were lower than those expected from simple 1st order kinetics and 1 : 1 inhibitor:catalyst stoichiometry. To rationalize the observed hypersensitivity, different kinetic scenarios were examined. For catalyst 2 and the Takemoto catalyst (6), the data is consistent with 2nd order dependency on catalyst concentration, suggesting that a mechanism involving only a single catalyst in the catalytic cycle is not operative. For catalyst 1, an alternative scenario involving 1st order in catalyst and catalyst poisoning at low concentrations of 1 could also rationalize the hypersensitivity. Interestingly, inhibition of catalysts 1 and 2 by halide salts led to significant loss of enantioselectivity, in contrast to the Takemoto catalyst 6 which was inhibited but with essentially no change in enantioselectivity. [ABSTRACT FROM AUTHOR]

The Bifidobacterium bifidum SAM-VI riboswitch undergoes dynamic conformational changes that modulate downstream gene expression. Traditional structural methods such as crystallography capture the bound conformation at high resolution, and additional efforts would reveal details from the dynamic transition. Here, we revealed a transcription-dependent conformation model for Bifidobacterium bifidum SAM-VI riboswitch. In this study, we combine small-angle X-ray scattering, chemical probing, and isothermal titration calorimetry to unveil the ligand-binding properties and conformational changes of the Bifidobacterium bifidum SAM-VI riboswitch and its variants. Our results suggest that the SAM-VI riboswitch contains a pre-organized ligand-binding pocket and stabilizes into the bound conformation upon binding to SAM. Whether the P1 stem formed and variations in length critically influence the conformational dynamics of the SAM-VI riboswitch. Our study provides the basis for artificially engineering the riboswitch by manipulating its peripheral sequences without modifying the SAM-binding core. [ABSTRACT FROM AUTHOR]

One colorimetric and one fluorescent‐indicator displacement assay (IDA) were studied to assess their interaction with methyl viologen (MV), an herbicide commercially known as paraquat. The host–guest complexes formed by cucurbit[7]uril (CB[7]) and the 4‐nitroaniline (NA) or Berberine (Be) dyes function as colorimetric‐ and fluorescent‐IDA for MV, respectively. The CB[7]@NA and CB[7]@Be complexes were characterized by UV–vis, fluorescence spectroscopy, and dynamic simulations. Isothermal titration calorimetry (ITC) experiments and dynamic simulations were employed to obtain thermodynamic data for the formation of the 1:1 inclusion complexes. Finally, NA is proposed as a reference colorimetric dye for determining CB[7]‐binding affinities using competitive host–guest titrations, and the fluorescent‐IDA as the best assay proposed, as it exhibits a high binding constant, a good detection limit of 2.7 × 10−6 mol L‐1, good linearity in the range from 4 × 10−6 mol L‐1 to 3.5 × 10−5 mol L‐1, in aqueous solution. [ABSTRACT FROM AUTHOR]

The aim of the research was to investigate and compare the interaction between flavanones (flavanone, 4-chloro-flavanone) with potential anticancer activity and selected cyclodextrins. Measurements were made using calorimetric (ITC, DSC) and spectrophotometric (UV-Vis spectroscopy, FT-IR, 1H NMR) methods. The increase in the solubility in aqueous medium caused by the complexation process was determined by the Higuchi-Connors method. As a result of the study, the stoichiometry and thermodynamics of the complexation reaction were determined. The formation of stable inclusion complexes at a 1:1 M ratio between flavanone and 4-chloroflavanone and the cyclodextrins selected for research was also confirmed. [ABSTRACT FROM AUTHOR]

Sensory adaptation in bacterial chemotaxis is mediated by posttranslational modifications of methyl‐accepting chemotaxis proteins (MCPs). In Escherichia coli, the adaptation proteins CheR and CheB tether to a conserved C‐terminal receptor pentapeptide. Here,we investigated the function of the pentapeptide motif (N/D)WE(E/N)F in Sinorhizobium meliloti chemotaxis. Isothermal titration calorimetry revealed stronger affinity of the pentapeptides to CheR and activated CheB relative to unmodified CheB. Strains with mutations of the conserved tryptophan in one or all four MCP pentapeptides resulted in a significant decrease or loss of chemotaxis to glycine betaine, lysine, and acetate, chemoattractants sensed by pentapeptide‐bearing McpX and pentapeptide‐lacking McpU and McpV, respectively. Importantly, we discovered that the pentapeptide mediates chemotaxis when fused to the C‐terminus of pentapeptide‐lacking chemoreceptors via a flexible linker. We propose that adaptational assistance and a threshold number of available sites enable the efficient docking of adaptation proteins to the chemosensory array. Altogether, these results demonstrate that S. meliloti effectively utilizes a pentapeptide‐dependent adaptation system with a minimal number of tethering units to assist pentapeptide‐lacking chemoreceptors and hypothesize that the higher abundance of CheR and CheB in S. meliloti compared to E. coli allows for ample recruitment of adaptation proteins to the chemosensory array. [ABSTRACT FROM AUTHOR]

Tetracyclines are a class of commonly used four‐ringed antibiotics. A series of DNA aptamers were recently obtained using the capture‐SELEX (systematic evolution of ligands by exponential enrichment) method to bind to oxytetracycline, and one of the aptamers can bind to a few other tetracycline antibiotics as well. Upon binding to the aptamers, the intrinsic fluorescence of tetracycline antibiotics can be enhanced. At least 10 different DNA aptamers were isolated from the previous selection experiment. In this work, a systematic characterization of these ten aptamers was performed. Each of these aptamers shows a different degree of fluorescence enhancement ranging from around 1‐fold to over 20‐fold. Fluorescence enhancement was boosted in the presence of Mg2+. Isothermal titration calorimetry (ITC) studies were done and showed a great variety in dissociation constant (Kd) from 62 nM to 1.6 μM. Steady‐state fluorescence spectroscopy and fluorescence lifetime studies showed a correlation between fluorescence lifetime and degree of fluorescence enhancement. A few aptamers showed slow binding kinetics, although no correlation was found between the kinetics of fluorescence change and degree of fluorescence enhancement. This is the first study of ten different aptamers for the same target, providing fundamental insights into aptamer binding and bioanalytical applications. [ABSTRACT FROM AUTHOR]

White-rot fungi differentially express laccases when they encounter aromatic compounds. However, the underlying mechanisms are still being explored. Here, proteomics analysis revealed that in addition to increased laccase activity, proteins involved in sphingolipid metabolism and toluene degradation as well as some cytochrome P450s (CYP450s) were differentially expressed and significantly enriched during 48 h of o-toluidine exposure, in Trametes hirsuta AH28-2. Two Zn2Cys6-type transcription factors (TFs), TH8421 and TH4300, were upregulated. Bioinformatics docking and isothermal titration calorimetry assays showed that each of them could bind directly to o-toluidine and another aromatic monomer, guaiacol. Binding to aromatic compounds promoted the formation of TH8421/TH4300 heterodimers. TH8421 and TH4300 silencing in T. hirsuta AH28-2 led to decreased transcriptional levels and activities of LacA and LacB upon o-toluidine and guaiacol exposure. EMSA and ChIP-qPCR analysis further showed that TH8421 and TH4300 bound directly with the promoter regions of lacA and lacB containing CGG or CCG motifs. Furthermore, the two TFs were involved in direct and positive regulation of the transcription of some CYP450s. Together, TH8421 and TH4300, two key regulators found in T. hirsuta AH28-2, function as heterodimers to simultaneously trigger the expression of downstream laccases and intracellular enzymes. Monomeric aromatic compounds act as ligands to promote heterodimer formation and enhance the transcriptional activities of the two TFs. [ABSTRACT FROM AUTHOR]

In the present work, the thermodynamic parameters for the adsorption of 2,4-dichlorophenoxyacetic acid (2,4-D), a herbicide, onto biomass-based activated carbon coated with magnetite nanoparticles (ACM) were estimated using a high-precision isothermal titration calorimeter (ITC). The obtained thermodynamic parameters, such as change in Gibb's free energy (ΔG), change in enthalpy (ΔH), and change in entropy (ΔS), were compared with the values computed applying conventional methods using equilibrium isotherm model parameters. The thermodynamic parameters suggested that the adsorption of 2,4-D onto ACM was spontaneous and exothermic. ITC proved to be an efficient experimental method in determining the thermodynamic parameters for the adsorption process with minimal adsorbent, adsorbate, and time consumption. The application of ITC can also be extended to determine the thermodynamic parameters of various solid–liquid interactions involved in different processes. Practical Applications: The 2,4-dichlorophenoxyacetic acid (2,4-D) is commonly used as a herbicide, and its occurrence is reported widely in aquatic bodies. Adsorption process using activated carbon impregnated with iron nanoparticles proved to be effective in removing these 2,4-D from the aquatic phase. In general, adsorption dynamics are demonstrated by isotherm, kinetics, and thermodynamic studies so that it can be extended to real-world applications. Conventionally, adsorption thermodynamic parameters are estimated from equilibrium isotherm constants. However, the determination of these parameters using the isotherm constants may not be accurate. Hence, sophisticated equipment such as isothermal titration calorimeter (ITC) can be used as an efficient tool to accurately compute the thermodynamic parameters in less time with minimal requirement of reagents. [ABSTRACT FROM AUTHOR]

Copyright of Farmacia is the property of Societatea de Stiinte Farmaceutice Romania and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Lewis acidic boron compounds are ubiquitous in chemistry due to their numerous applications, yet tuning and optimizing their properties towards different purposes is still a challenging field of research. In this work, the boron‐based Lewis acid B[OTeF3(C6F5)2]3 was synthesized by reaction of the teflate derivative HOTeF3(C6F5)2 with BCl3 or BCl3 ⋅ SMe2. This new compound presents a remarkably high thermal stability up to 300 °C, as well as one of the most sterically encumbered boron centres known in the literature. Theoretical and experimental methods revealed that B[OTeF3(C6F5)2]3 exhibits a comparable Lewis acidity to that of the well‐known B(C6F5)3. The affinity of B[OTeF3(C6F5)2]3 towards pyridine was accessed by Isothermal Titration Calorimetry (ITC) and compared to that of B(OTeF5)3 and B(C6F5)3. The ligand‐transfer reactivity of this new boron compound towards different fluorides was demonstrated by the formation of an anionic Au(III) complex and a hypervalent iodine(III) species. [ABSTRACT FROM AUTHOR]

The human cationic antimicrobial protein (hCAP) corresponding to the overlapping sequences of 151–162 of hCAP named KR-12 peptide is the smallest portion of the only type of human Cathelicidin, which has been shown to be modifiable into a more effective antimicrobial. In this study, an in silico analysis, supported by potentiometric titration and isothermal titration calorimetry techniques, was performed to identify potential Cu(II) binding sites of KR-12. The analysis of the presented data at the given theoretical level (GFN2-xTB/ALPB) revealed which peptide chain fragments are involved in the most favourable KR-12–Cu(II) binding mode. Based on a quantum chemical approach, the most favourable coordination modes of Cu(II) to peptides are proposed together with the discussion of the chemical nature of the interactions. The presented results demonstrated that KR-12 interacts with metal ions mostly via the main chain's oxygen atoms; however, the two types of amino acids that are expected to be vital for the interaction of Cu(II) are D (aspartic acid) and R29 (arginine). It was demonstrated that in order to explain the complexity of the interaction process in peptide–metal ion systems, the use of theoretical methods is sometimes necessary to explain the details of the experimental results and provide an in-depth understanding of these dynamic systems. [ABSTRACT FROM AUTHOR]

Ecological restoration of bauxite residue has received extensive attention, and organic matter plays a crucial role in the soil formation process of bauxite residue. However, the interaction between organic matter and alkaline minerals in bauxite residue is not well understood. In this work, molecular spectroscopic techniques combined with isothermal titration calorimetry (ITC) were employed to investigate the interactions between humic acid (HA) and four representative alkaline minerals in bauxite residue (calcite, garnet, sodalite, and cancrinite). The results show that the adsorption processes of HA onto calcite and garnet were primarily governed by monolayer surface adsorption and controlled by surface reactions, which were different for sodalite and cancrinite. Both garnet and cancrinite had strong binding affinities with fluorescent HA, while cancrinite only bound with a small fraction of HA. In contrast, the bindings of calcite and sodalite with fluorescent HA were weak. The ITC results indicate distinct thermodynamic properties of different alkaline minerals in the interaction with HA. The molar enthalpy of calcite was − 45.88 kJ/mol, which was much higher than those of garnet, sodalite, and cancrinite, suggesting that calcite exhibited a relatively uniform interaction mechanism with HA dominated by enthalpy change, while the others showed heterogeneous entropy-driven mechanisms. The findings contribute to a better understanding on the microscale connections between organic matter and alkaline minerals in bauxite residue. [ABSTRACT FROM AUTHOR]

A new concept for the regioselective synthesis of Hamilton‐receptor and cyanurate‐functionalized oligo adducts of the fullerene C60 was developed. Based on an in‐situ deprotection and click‐post‐functionalization approach with novel azido precursors, the corresponding fullerene hexakis‐adducts with octahedral addition patterns and up to twelve Hamilton‐receptor/cyanurate moieties surrounding the fullerene sphere were synthesized. The versatility of this approach was further demonstrated by the synthesis of Hamilton‐receptor/cyanurate functionalized fullerene mono‐adducts, which are not accessible by direct cyclopropanation. Several fullerene target compounds were purified by simple washing procedures of the solid crude reaction mixture without the need for chromatography. The resulting fullerene mono‐ and hexakis‐adducts were fully characterized and their supramolecular properties were investigated by NMR‐spectroscopy and isothermal titration calorimetry (ITC). [ABSTRACT FROM AUTHOR]

We study the hyperactivation of α‐chymotrypsin (α‐ChT) using the acrylate polymer poly(2‐carboxyethyl) acrylate (PCEA) in comparison to the commonly used poly(acrylic acid) (PAA). The polymers are added during the enzymatic cleavage reaction of the substrate N‐glycyl‐L‐phenylalanine p‐nitroanilide (GPNA). Enzyme activity assays reveal a pronounced enzyme hyperactivation capacity of PCEA, which reaches up to 950 % activity enhancement, and is significantly enhanced to PAA (revealing an activity enhancement of approx. 450 %). In a combined experimental and computational study, we investigate α‐ChT/polymer interactions to elucidate the hyperactivation mechanism of the enzyme. Isothermal titration calorimetry reveals a pronounced complexation between the polymer and the enzyme. Docking simulations reveal that binding of polymers significantly improves the binding affinity of GPNA to α‐ChT. Notably, a higher binding affinity is found for the α‐ChT/PCEA compared to the α‐ChT/PAA complex. Further molecular dynamics (MD) simulations reveal changes in the size of the active site in the enzyme/polymer complexes, with PCEA inducing a more pronounced alteration compared to PAA, facilitating an easier access for the substrate to the active site of α‐ChT. [ABSTRACT FROM AUTHOR]

Incidences of drug-resistant tuberculosis have become common and are rising at an alarming rate. Aminoacyl t-RNA synthetase has been validated as a newer target against Mycobacterium tuberculosis. Leucyl t-RNA synthetase (LeuRS) is ubiquitously found in all organisms and regulates transcription, protein synthesis, mitochondrial RNA cleavage, and proofreading of matured t-RNA. Leucyl t-RNA synthetase promotes growth and development and is the key enzyme needed for biofilm formation in Mycobacterium. Inhibition of this enzyme could restrict the growth and development of the mycobacterial population. A database consisting of 2734 drug-like molecules was screened against leucyl t-RNA synthetase enzymes through virtual screening. Based on the docking scores and MMGBSA energy values, the top three compounds were selected for molecular dynamics simulation. The druggable nature of the top three hits was confirmed by predicting their pharmacokinetic parameters. The top three hits—compounds 1035 (ZINC000001543916), 1054 (ZINC000001554197), and 2077 (ZINC000008214483)—were evaluated for their binding affinity toward leucyl t-RNA synthetase by an isothermal titration calorimetry study. The inhibitory activity of these compounds was tested against antimycobacterial activity, biofilm formation, and LeuRS gene expression potential. Compound 1054 (Macimorelin) was found to be the most potent molecule, with better antimycobacterial activity, enzyme binding affinity, and significant inhibition of biofilm formation, as well as inhibition of the LeuRS gene expression. Compound 1054, the top hit compound, has the potential to be used as a lead to develop successful leucyl t-RNA synthetase inhibitors. [ABSTRACT FROM AUTHOR]

Forkhead box P2 (FOXP2) regulates expression of various genes and is associated with language, speech and neural development as well as cancer. Since there may be a putative link between sex and language and because transcription factors rarely function in isolation, this study aims to investigate whether FOXP2 directly associates with oestrogen receptor α (ER1), a nuclear receptor responsible for sexual differentiation that is also associated with cancer. Isothermal titration calorimetry and fluorescence anisotropy were used to investigate the interaction between the DNA-binding forkhead domain (FHD) of FOXP2, the N-terminal region (NT) of FOXP2, and the ligand-binding domain (LBD) of ER1. ER1 LBD does not interact with FOXP2 NT but associates with apo-FOXP2 FHD in an enthalpically favourable manner. The affinity of this interaction is inversely correlated to the salt concentration. Additionally, FOXP2 FHD that is bound to ER1 LBD, has reduced ability to interact with its cognate DNA. This research identifies a novel interaction between ER1 LBD and FOXP2 FHD and shows that the interaction is regulated by salt. Moreover, FOXP2 FHD cannot bind to both ER1 LBD and DNA simultaneously, suggesting that this interaction could be involved in regulating the transcriptional pathway of FOXP2 should the interaction be found in vivo. This study could serve as a foundation for uncovering the basis of sexual dimorphism in speech and language development and related disorders and potentially offers an alternate for targeted cancer therapies. • A novel interaction is identified in vitro between FOXP2 FHD and ER1 LBD. • This interaction has both an electrostatic and hydrophobic interface and involves the DNA-binding Helix 3 of FOXP2 FHD. • The ligand binding region and the LXXLL-binding AF-2 region of ER1 LBD is not involved in the protein-protein interaction. • FOXP2 FHD can bind either to DNA or to ER1 but not to both simultaneously. • The identified FOXP2-ER1 interaction could be involved in regulating FOXP2's transcriptional activity. [ABSTRACT FROM AUTHOR]

Proteins containing DM9 motifs, which were originally identified in the Drosophila melanogaster genome, are widely distributed in various organisms and are assumed to be involved in their innate immune response. In this study, we produced a recombinant protein of CG13321 (rCG13321) from D. melanogaster , which consists of four DM9 motifs, in Escherichia coli cells. In affinity chromatography using a mannose-immobilized column, rCG13321 exhibited mannose-binding ability and was separated into high-affinity and low-affinity fractions, named HA and LA, respectively, based on its binding ability to the column. In addition to having a higher affinity for the column, HA exhibited self-oligomerization ability, suggesting slight differences in tertiary structure. Both LA and HA showed hemagglutinating activity and were able to agglutinate an oligomannose-containing dendrimer, indicating that they have multiple carbohydrate-binding sites. Glycan array analysis suggested that rCG13321 primarily recognizes d -mannose and d -rhamnose through hydrogen bonding with the 2-, 3- and 4-hydroxy groups. Isothermal titration calorimetry demonstrated that rCG13321 has a comparable affinity to typical lectins. These findings suggest that CG13321 functions as a carbohydrate-binding protein or lectin that recognizes mannose and related carbohydrate-containing molecules on the surface of foreign organisms as a pattern recognition molecule. [ABSTRACT FROM AUTHOR]

Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of Desulfitobacterium hafniense CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the "half-of-the-sites" ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a "two non-interacting pairs of interacting regulatory sites" concept in CBS-PPase regulation. [ABSTRACT FROM AUTHOR]

Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI's unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells. [ABSTRACT FROM AUTHOR]