Signal integration and adaptive sensory diversity tuning in Escherichia coli chemotaxis.

In uncertain environments, phenotypic diversity can be advantageous for survival. However, as the environmental uncertainty decreases, the relative advantage of having diverse phenotypes decreases. Here, we show how populations of E. coli integrate multiple chemical signals to adjust sensory diversity in response to changes in the prevalence of each ligand in the environment. Measuring kinase activity in single cells, we quantified the sensitivity distribution to various chemoattractants in different mixtures of background stimuli. We found that when ligands bind uncompetitively, the population tunes sensory diversity to each signal independently, decreasing diversity when the signal's ambient concentration increases. However, among competitive ligands, the population can only decrease sensory diversity one ligand at a time. Mathematical modeling suggests that sensory diversity tuning benefits E. coli populations by modulating how many cells are committed to tracking each signal proportionally as their prevalence changes.

Bivalent affinity binding-inspired PPARγ immobilization with selective conformation and improved ligand-binding activity.

Functional protein immobilization forms the basis for bio-detections. A series of one-point, site-specific immobilization methods have been developed, however, it still remains as a challenge how to avoid the proteins to move in all directions as well as conveniently regenerate the bio-devices. Herein, we have developed a bivalent affinity binding-inspired method for PPARγ immobilization using DNA aptamer and nickel-nitrilotriacetic acid (Ni2+-NTA) chelation. The specific DNA aptamer (Apt 2) was selected by an on-column systematic evolution of ligands by exponential enrichment (SELEX) method with affinity of (1.57 ± 0.15) × 105 M-1, determined by isothermal titration calorimetry (ITC). Apt 2 and nickel-nitrilotriacetic acid (Ni2+-NTA) were modified on macroporous silica gels via L-α-allylglycine as a linker. They respectively interacted with PPARγ and 6×His tag via bivalent affinity binding for the receptor immobilization. After comprehensive surface characterization, PPARγ was proved to be successful immobilized. Chromatographic studies revealed that the immobilized PPARγ has conformation selectivity, which discriminated agonist and antagonist of the receptor. Ligand-binding parameters (affinity and rate constant) of four agonists (rosiglitazone, pioglitazone, troglitazone, and magnolol) with PPARγ were determined. Troglitazone showed the lowest dissociation rate constant. The binding affinities (3.28 × 107, 1.91 × 106, 2.25 × 107, and 2.43 × 107 M-1) were highly consistent with the data obtained using purified receptor in solution (2.16 × 107, 4.52 × 106, 1.20 × 107, and 1.56 × 107 M-1), offering reliable bio-detection method for PPARγ and its ligands. Due to the biocompatibility of nuclear receptor with DNA, it is conceivable that the bivalent affinity-based method will be a general method for the immobilization of other nuclear receptors, which may provide selective conformation and improved ligand-binding activity for the receptors.

Rapid Determination of Kinetic Constants for Slow-Binding Inhibitors and Inactivators of Human Histone Deacetylase 8.

The kinetics and mechanism of drug binding to its target are critical to pharmacological efficacy. A high throughput (HTS) screen often results in hundreds of hits, of which usually only simple IC50 values are determined during reconfirmation. However, kinetic parameters such as residence time for reversible inhibitors and the kinact/KI ratio, which is the critical measure for evaluating covalent inactivators, are early predictive measures to assess the chances of success of the hits in the clinic. Using the promising cancer target human histone deacetylase 8 as an example, we present a robust method that calculates concentration-dependent apparent rate constants for the inhibition or inactivation of HDAC8 from dose-response curves recorded after different pre-incubation times. With these data, hit compounds can be classified according to their mechanism of action, and the relevant kinetic parameters can be calculated in a highly parallel fashion. HDAC8 inhibitors with known modes of action were correctly assigned to their mechanism, and the binding mechanisms of some hits from an internal HDAC8 screening campaign were newly determined. The oxonitriles SVE04 and SVE27 were classified as fast reversible HDAC8 inhibitors with moderate time-constant IC50 values of 4.2 and 2.6 µM, respectively. The hit compound TJ-19-24 and SAH03 behave like slow two-step inactivators or reversible inhibitors, with a very low reverse isomerization rate.

AI-based prediction of protein-ligand binding affinity and discovery of potential natural product inhibitors against ERK2.

Determination of protein-ligand binding affinity (PLA) is a key technological tool in hit discovery and lead optimization, which is critical to the drug development process. PLA can be determined directly by experimental methods, but it is time-consuming and costly. In recent years, deep learning has been widely applied to PLA prediction, the key of which lies in the comprehensive and accurate representation of proteins and ligands. In this study, we proposed a multi-modal deep learning model based on the early fusion strategy, called DeepLIP, to improve PLA prediction by integrating multi-level information, and further used it for virtual screening of extracellular signal-regulated protein kinase 2 (ERK2), an ideal target for cancer treatment. Experimental results from model evaluation showed that DeepLIP achieved superior performance compared to state-of-the-art methods on the widely used benchmark dataset. In addition, by combining previously developed machine learning models and molecular dynamics simulation, we screened three novel hits from a drug-like natural product library. These compounds not only had favorable physicochemical properties, but also bound stably to the target protein. We believe they have the potential to serve as starting molecules for the development of ERK2 inhibitors.

Allosteric modulation of serotonin and dopamine transporters: New insights from computations and experiments.

Human monoamine transporters (MATs) are critical to regulating monoaminergic neurotransmission by translocating their substrates from the synaptic space back into the presynaptic neurons. As such, their primary substrate binding site S1 has been targeted by a wide range of compounds for treating neuropsychiatric and neurodegenerative disorders including depression, ADHD, neuropathic pain, and anxiety disorders. We present here a comparative study of the structural dynamics and ligand-binding properties of two MATs, dopamine transporter (DAT) and serotonin transporter (SERT), with focus on the allosteric modulation of their transport function by drugs or substrates that consistently bind a secondary site S2, proposed to serve as an allosteric site. Our systematic analysis of the conformational space and dynamics of a dataset of 50 structures resolved for DAT and SERT in the presence of one or more ligands/drugs reveals the specific residues playing a consistent role in coordinating the small molecules bound to subsites S2-I and S2-II within S2, such as R476 and Y481 in dDAT and E494, P561, and F556 in hSERT. Further analysis reveals how DAT and SERT differ in their two principal modes of structural changes, PC1 and PC2. Notably, PC1 underlies the transition between outward- and inward-facing states of the transporters as well as their gating; whereas PC2 supports the rearrangements of TM helices near the S2 site. Finally, the examination of cross-correlations between structural elements lining the respective sites S1 and S2 point to the crucial role of coupled motions between TM6a and TM10. In particular, we note the involvement of hSERT residues F335 and G338, and E493-E494-T497 belonging to these two respective helices, in establishing the allosteric communication between S1 and S2. These results help understand the molecular basis of the action of drugs that bind to the S2 site of DAT or SERT. They also provide a basis for designing allosteric modulators that may provide better control of specific interactions and cellular pathways, rather than indiscriminately inhibiting the transporter by targeting its orthosteric site.

Machine Learning in Enhancing Protein Binding Sites Predictions - What Has Changed Since Then?

Accurate identification of protein binding sites is pivotal for understanding molecular interactions and facilitating drug discovery efforts. However, the dynamic nature of proteinligand interactions presents a formidable challenge, necessitating innovative approaches to bridge the gap between theoretical predictions and experimental realities. This review explores the challenges and recent advancements in protein binding site prediction. Specifically, we highlight the integration of molecular dynamics simulations, machine learning, and deep learning techniques to capture the dynamic and complex nature of protein-ligand interactions. Additionally, we discuss the importance of integrating experimental data, such as structural information and biochemical assays, to enhance prediction accuracy and reliability. By navigating the intersection of classical and the onset of machine learning and deep learning approaches, we aim to provide insights into current state-of-the-art techniques and chart a course for future protein binding site prediction advancements. Ultimately, these efforts could unravel the mysteries of protein-ligand interactions and accelerate drug discovery endeavors.

In silico bioprospecting of receptors associated with the mechanism of action of Rondonin, an antifungal peptide from spider Acanthoscurria rondoniae haemolymph.

Multiple drug-resistant fungal species are associated with the development of diseases. Thus, more efficient drugs for the treatment of these aetiological agents are needed. Rondonin is a peptide isolated from the haemolymph of the spider Acanthoscurria rondoniae. Previous studies have shown that this peptide has antifungal activity against Candida sp. and Trichosporon sp. strains, acting on their genetic material. However, the molecular targets involved in its biological activity have not yet been described. Bioinformatics tools were used to determine the possible targets involved in the biological activity of Rondonin. The PharmMapper server was used to search for microorganismal targets of Rondonin. The PatchDock server was used to perform the molecular docking. UCSF Chimera software was used to evaluate these intermolecular interactions. In addition, the I-TASSER server was used to predict the target ligand sites. Then, these predictions were contrasted with the sites previously described in the literature. Molecular dynamics simulations were conducted for two promising complexes identified from the docking analysis. Rondonin demonstrated consistency with the ligand sites of the following targets: outer membrane proteins F (id: 1MPF) and A (id: 1QJP), which are responsible for facilitating the passage of small molecules through the plasma membrane; the subunit of the flavoprotein fumarate reductase (id: 1D4E), which is involved in the metabolism of nitrogenous bases; and the ATP-dependent Holliday DNA helicase junction (id: 1IN4), which is associated with histone proteins that package genetic material. Additionally, the molecular dynamics results indicated the stability of the interaction of Rondonin with 1MPF and 1IN4 during a 10 ns simulation. These interactions corroborate with previous in vitro studies on Rondonin, which acts on fungal genetic material without causing plasma membrane rupture. Therefore, the bioprospecting methods used in this research were considered satisfactory since they were consistent with previous results obtained via in vitro experimentation. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00224-1.

LDLR c.89_92dup: a novel frameshift variation in familial hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) is a common inherited metabolic disease that causes premature atherosclerosis, cardiovascular disease, and even death at a young age. Approximately 95% of FH-causing genetic variants that have been identified are in the LDLR gene. However, only 10% of the FH population worldwide has been diagnosed and adequately treated, due to the existence of numerous unidentified variants, uncertainties in the pathogenicity scoring of many variants, and a substantial number of individuals lacking access to genetic testing. OBJECTIVE: The aim of this study was to identify a novel variant in the LDLR gene that causes FH in a Chinese family, thereby expanding the spectrum of FH-causing variants. METHODS: Patients were recruited from Beijing Anzhen Hospital, Capital Medical University. FH diagnosis was made according to the Dutch Lipid Clinical Network (DLCN) criteria. Whole-exome sequencing (WES) was conducted to identify the FH-causing variant in the proband, and amplicon sequencing was used to verify the variant in his family members. RESULTS: A three-generation Chinese family was recruited, and two FH patients were clinically diagnosed, both without known FH-causing variants. These two FH patients and another possible patient carried a novel variant, NC_000019.9(NM_000527.5):c.89_92dup (NP_000518.1:p.Phe32Argfs*21), in the ligand-binding domain of the low-density lipoprotein (LDL) receptor that led to a frameshift. The FH adults in the family showed severe clinical symptoms and statin therapy resistance. CONCLUSION: This study identified a novel pathogenic LDLR variant, c.89_92dup, associated with severe FH clinical manifestations and statin therapy resistance.

Novel [68Ga/177Lu]Ga/Lu-AZ-093 as PSMA-Targeting Agent for Diagnosis and Radiotherapy.

Prostate-specific membrane antigen (PSMA) overexpressed in prostate cancer cells can serve as a target for imaging and radioligand therapy (RLT). Previously, [68Ga]Ga-P16-093, containing a Ga(III) chelator, N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC), displayed excellent PSMA-targeting properties and showed a high tumor uptake and retention useful for diagnosis in prostate cancer patients. Recently, [177Lu]Lu-PSMA-617 has been approved by the U.S. food and drug administration (FDA) for the treatment of prostate cancer patients. Derivatives of PSMA-093 using AAZTA (6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid), as the chelator, were designed as alternative agents forming complexes with both diagnostic and therapeutic radiometals, such as gallium-68 (log K = 22.18) or lutetium-177 (log K = 21.85). The aim of this study is to evaluate AAZTA-Gly-O-(methylcarboxy)-Tyr-Phe-Lys-NH-CO-NH-Glu (designated as AZ-093, 1) leading to a gallium-68/lutetium-177 theranostic pair as potential PSMA targeting agents. Synthesis of the desired precursor, AZ-093, 1, was effectively accomplished. Labeling with either [68Ga]GaCl3 or [177Lu]LuCl3 in a sodium acetate buffer solution (pH 4-5) at 50 °C in 5 to 15 min produced either [68Ga]Ga-1 or [177Lu]Lu-1 with high yields and excellent radiochemical purities. Results of in vitro binding studies, cell uptake, and retention (using PSMA-positive prostate carcinoma cells line, 22Rv1-FOLH1-oe) were comparable to that of [68Ga]Ga-P16-093 and [177Lu]Lu-PSMA-617, respectively. Specific cellular uptake was determined with or without the competitive blocking agent (2 µM of "cold" PSMA-11). Cellular binding and internalization showed a time-dependent increase over 2 h at 37 °C in the PSMA-positive cells. The cell uptakes were completely blocked by the "cold" PSMA-11 suggesting that they are competing for the same PSMA binding sites. In the mouse model with implanted PSMA-positive tumor cells, both [68Ga]Ga-1 and [177Lu]Lu-1 displayed excellent uptake and retention in the tumor. Results indicate that [68Ga]Ga/[177Lu]Lu-1 (68Ga]Ga/[177Lu]Lu-AZ-093) is potentially useful as PSMA-targeting agent for both diagnosis and radiotherapy of prostate cancer.

Structure and the Anticancer Activity of Vitamin D Receptor Agonists.

Vitamin D is a group of seco-steroidal fat-soluble compounds. The two basic forms, vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol), do not have biological activity. They are converted in the body by a two-step enzymatic hydroxylation into biologically active forms, 1α,25-dihydroxyvitamin D2 [ercalcitriol, 1,25(OH)2D2] and 1α,25-dihydroxyvitamin D3 [calcitriol, 1,25(OH)2D3], which act as classical steroid hormones. 1,25(OH)2D3 exerts most of its physiological functions by binding to the nuclear vitamin D receptor (VDR), which is present in most body tissues to provide support to a broad range of physiological processes. Vitamin D-liganded VDR controls the expression of many genes. High levels of 1,25(OH)2D3 cause an increase in calcium in the blood, which can lead to harmful hypercalcemia. Several analogs of 1,25(OH)2D3 and 1,25(OH)2D2 have been designed and synthesized with the aim of developing compounds that have a specific therapeutic function, for example, with potent anticancer activity and a reduced toxic calcemic effect. Particular structural modifications to vitamin D analogs have led to increased anticancer activity and reduced calcemic action with the prospect of extending work to provide future innovative therapies.

From X-ray crystallographic structure to intrinsic thermodynamics of protein-ligand binding using carbonic anhydrase isozymes as a model system.

Carbonic anhydrase (CA) was among the first proteins whose X-ray crystal structure was solved to atomic resolution. CA proteins have essentially the same fold and similar active centers that differ in only several amino acids. Primary sulfonamides are well defined, strong and specific binders of CA. However, minor variations in chemical structure can significantly alter their binding properties. Over 1000 sulfonamides have been designed, synthesized and evaluated to understand the correlations between the structure and thermodynamics of their binding to the human CA isozyme family. Compound binding was determined by several binding assays: fluorescence-based thermal shift assay, stopped-flow enzyme activity inhibition assay, isothermal titration calorimetry and competition assay for enzyme expressed on cancer cell surfaces. All assays have advantages and limitations but are necessary for deeper characterization of these protein-ligand interactions. Here, the concept and importance of intrinsic binding thermodynamics is emphasized and the role of structure-thermodynamics correlations for the novel inhibitors of CA IX is discussed - an isozyme that is overexpressed in solid hypoxic tumors, and thus these inhibitors may serve as anticancer drugs. The abundant structural and thermodynamic data are assembled into the Protein-Ligand Binding Database to understand general protein-ligand recognition principles that could be used in drug discovery.

Probing G-quadruplex-ligand binding using DNA intrinsic fluorescence.

G-quadruplexes (G4s) are helical four-stranded nucleic acid structures that can form in guanine-rich sequences, which are mostly found in functional cellular regions, such as telomeres, promoters, and DNA replication origins. Great efforts are being made to target these structures towards the development of specific small molecule G4 binders for novel anti-cancer, neurological, and viral therapies. Here, we introduce an optical assay based on quenching of the intrinsic fluorescence of DNA G-quadruplexes for assessing and comparing the G4 binding affinity of various small molecule ligands in solutions. We show that the approach allows direct quantification of ligand binding to distinctive G4 topologies. We believe that this method will facilitate quick and reliable evaluation of small molecule G4 ligands and support their development.

Q-BioLiP: A Comprehensive Resource for Quaternary Structure-based Protein-ligand Interactions.

Since its establishment in 2013, BioLiP has become one of the widely used resources for protein-ligand interactions. Nevertheless, several known issues occurred with it over the past decade. For example, the protein-ligand interactions are represented in the form of single chain-based tertiary structures, which may be inappropriate as many interactions involve multiple protein chains (known as quaternary structures). We sought to address these issues, resulting in Q-BioLiP, a comprehensive resource for quaternary structure-based protein-ligand interactions. The major features of Q-BioLiP include: (1) representing protein structures in the form of quaternary structures rather than single chain-based tertiary structures; (2) pairing DNA/RNA chains properly rather than separation; (3) providing both experimental and predicted binding affinities; (4) retaining both biologically relevant and irrelevant interactions to alleviate the wrong justification of ligands' biological relevance; and (5) developing a new quaternary structure-based algorithm for the modelling of protein-ligand complex structure. With these new features, Q-BioLiP is expected to be a valuable resource for studying biomolecule interactions, including protein-small molecule interaction, protein-metal ion interaction, protein-peptide interaction, protein-protein interaction, protein-DNA/RNA interaction, and RNA-small molecule interaction. Q-BioLiP is freely available at https://yanglab.qd.sdu.edu.cn/Q-BioLiP/.

Molecular basis for the activation of PAF receptor by PAF.

Platelet-activating factor (PAF) is a potent phospholipid mediator crucial in multiple inflammatory and immune responses through binding and activating the PAF receptor (PAFR). However, drug development targeting the PAFR has been limited, partly due to an incomplete understanding of its activation mechanism. Here, we present a 2.9-Å structure of the PAF-bound PAFR-Gi complex. Structural and mutagenesis analyses unveil a specific binding mode of PAF, with the choline head forming cation-π interactions within PAFR hydrophobic pocket, while the alkyl tail penetrates deeply into an aromatic cleft between TM4 and TM5. Binding of PAF modulates conformational changes in key motifs of PAFR, triggering the outward movement of TM6, TM7, and helix 8 for G protein coupling. Molecular dynamics simulation suggests a membrane-side pathway for PAF entry into PAFR via the TM4-TM5 cavity. By providing molecular insights into PAFR signaling, this work contributes a foundation for developing therapeutic interventions targeting PAF signal axis.

The fully reduced terminal oxidase bd-I isolated from Escherichia coli binds cyanide.

Cytochrome bd-I from Escherichia coli belongs to the superfamily of prokaryotic bd-type oxygen reductases. It contains three hemes, b558, b595 and d, and couples oxidation of quinol by dioxygen with the generation of a proton-motive force. The enzyme exhibits resistance to various stressors and is considered as a target protein for next-generation antimicrobials. By using electronic absorption and MCD spectroscopy, this work shows that cyanide binds to heme d2+ in the isolated fully reduced cytochrome bd-I. Cyanide-induced difference absorption spectra display changes near the heme d2+ α-band, a minimum at 633 nm and a maximum around 600 nm, and a W-shaped response in the Soret region. Apparent dissociation constant (Kd) of the cyanide complex of heme d2+ is ∼0.052 M. Kinetics of cyanide binding is monophasic, indicating the presence of a single ligand binding site in the enzyme. Consistently, MCD data show that cyanide binds to heme d2+ but not to b5582+ or b5952+. This agrees with the published structural data that the enzyme's active site is not a di-heme site. The observed rate of binding (kobs) increases as the concentration of cyanide is increased, giving a second-order rate constant (kon) of ∼0.1 M-1 s-1.

Gas6 and Protein S Ligands Cooperate to Regulate MerTK Rhythmic Activity Required for Circadian Retinal Phagocytosis.

Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an outstanding contribution from the team of Prof. Greg Lemke. MerTK activity is implicated in a wide variety of functions involving the elimination of apoptotic cells and has recently been linked to cancers, auto-immune diseases, and atherosclerosis/stroke. In the retina, MerTK is required for the circadian phagocytosis of oxidized photoreceptor outer segments by the retinal-pigment epithelial cells, a function crucial for the long-term maintenance of vision. We previously showed that MerTK ligands carry the opposite role in vitro, with Gas6 inhibiting the internalization of photoreceptor outer segments while Protein S acts conversely. Using site-directed mutagenesis and ligand-stimulated phagocytosis assays on transfected cells, we presently demonstrate, for the first time, that Gas6 and Protein S recognize different amino acids on MerTK Ig-like domains. In addition, MerTK's function in retinal-pigment epithelial cells is rhythmic and might thus rely on the respective stoichiometry of both ligands at different times of the day. Accordingly, we show that ligand bioavailability varies during the circadian cycle using RT-qPCR and immunoblots on retinal and retinal-pigment epithelial samples from control and beta5 integrin knockout mice where retinal phagocytosis is arrhythmic. Taken together, our results suggest that Gas6 and Protein S might both contribute to refine the acute regulation of MerTK in time for the daily phagocytic peak.

Unique challenges required reassessment and alterations to critical reagents to rescue a neutralizing antibody assay.

Aim: To redevelop a neutralizing antibody (NAb) assay to be much more drug tolerant, have a large dynamic range and have high inhibition when using high levels of positive control (PC). Materials & methods: Early assay data suggested that typical biotin labeling of the capture reagent (Drug 1, produced in a human cell line) was blocking it from binding with the PC or the detection target, and that the detection target was out competing the PC. Methodical biotin labeling experiments were performed at several challenge ratios and an Fc linker was added to the detection target. Results & conclusion: A larger dynamic range, high inhibition and higher drug tolerance were achieved by adding an acid dissociation step to the assay, performing atypical biotin labeling of Drug 1 and switching to a detection target that contained an Fc linker to increase steric hinderance and decrease its binding affinity to Drug 1.

Many of the drugs available today are produced by a living organism and these are called biologics. Biologics are larger than chemical drugs and the human body can detect them as foreign and create antibodies against them. This is called immunogenicity. When the antibodies created against the biologic blocks the drug's ability to work correctly, they are called neutralizing antibodies (NAbs). Testing for NAbs is one of the requirements of regulatory agencies for biologics. Here we describe challenges encountered developing an assay to test for NAbs against a biologic.

Interfacial Interactions between Nanoplastics and Biological Systems: toward an Atomic and Molecular Understanding of Plastics-Driven Biological Dyshomeostasis.

Micro- and nano-plastics (NPs) are found in human milk, blood, tissues, and organs and associate with aberrant health outcomes including inflammation, genotoxicity, developmental disorders, onset of chronic diseases, and autoimmune disorders. Yet, interfacial interactions between plastics and biomolecular systems remain underexplored. Here, we have examined experimentally, in vitro, in vivo, and by computation, the impact of polystyrene (PS) NPs on a host of biomolecular systems and assemblies. Our results reveal that PS NPs essentially abolished the helix-content of the milk protein ß-lactoglobulin (BLG) in a dose-dependent manner. Helix loss is corelated with the near stoichiometric formation of ß-sheet elements in the protein. Structural alterations in BLG are also likely responsible for the nanoparticle-dependent attrition in binding affinity and weaker on-rate constant of retinol, its physiological ligand (compromising its nutritional role). PS NP-driven helix-to-sheet conversion was also observed in the amyloid-forming trajectory of hen egg-white lysozyme (accelerated fibril formation and reduced helical content in fibrils). Caenorhabditis elegans exposed to PS NPs exhibited a decrease in the fluorescence of green fluorescent protein-tagged dopaminergic neurons and locomotory deficits (akin to the neurotoxin paraquat exposure). Finally, in silico analyses revealed that the most favorable PS/BLG docking score and binding energies corresponded to a pose near the hydrophobic ligand binding pocket (calyx) of the protein where the NP fragment was found to make nonpolar contacts with side-chain residues via the hydrophobic effect and van der Waals forces, compromising side chain/retinol contacts. Binding energetics indicate that PS/BLG interactions destabilize the binding of retinol to the protein and can potentially displace retinol from the calyx region of BLG, thereby impairing its biological function. Collectively, the experimental and high-resolution in silico data provide new insights into the mechanism(s) by which PS NPs corrupt the bimolecular structure and function, induce amyloidosis and onset neuronal injury, and drive aberrant physiological and behavioral outcomes.

Spectroscopic, electrochemical, and kinetic trends in Fe(III)-thiolate disproportionation near physiologic pH.

In addition to its primary oxygen-atom-transfer function, cysteamine dioxygenase (ADO) exhibits a relatively understudied anaerobic disproportionation reaction (ADO-Fe(III)-SR → ADO-Fe(II) + ½ RSSR) with its native substrates. Inspired by ADO disproportionation reactivity, we employ [Fe(tacn)Cl3] (tacn = 1,4,7-triazacyclononane) as a precursor for generating Fe(III)-thiolate model complexes in buffered aqueous media. A series of Fe(III)-thiolate model complexes are generated in situ using aqueous [Fe(tacn)Cl3] and thiol-containing ligands cysteamine, penicillamine, mercaptopropionate, cysteine, cysteine methyl ester, N-acetylcysteine, and N-acetylcysteine methyl ester. We observe trends in UV-Vis and electron paramagnetic resonance (EPR) spectra, disproportionation rate constants, and cathodic peak potentials as a function of thiol ligand. These trends will be useful in rationalizing substrate-dependent Fe(III)-thiolate disproportionation reactions in metalloenzymes.

Genomic analysis of Salmonella bacteriophages revealed multiple endolysin ORFs and importance of ligand-binding site of receptor-binding protein.

Salmonella is a prevalent foodborne pathogen causing millions of global cases annually. Antimicrobial resistance is a growing public health concern, leading to search for alternatives like bacteriophages. A total of 97 bacteriophages, isolated from cattle farms (n = 48), poultry farms (n = 37), and wastewater (n = 5) samples in Türkiye, were subjected to host-range analysis using 36 Salmonella isolates with 18 different serotypes. The broadest host range belonged to an Infantis phage (MET P1-091), lysing 28 hosts. A total of 10 phages with the widest host range underwent further analysis, revealing seven unique genomes (32-243 kb), including a jumbophage (>200 kb). Except for one with lysogenic properties, none of them harbored virulence or antibiotic resistance genes, making them potential Salmonella reducers in different environments. Examining open reading frames (ORFs) of endolysin enzymes revealed surprising findings: five of seven unique genomes contained multiple endolysin ORFs. Despite sharing same endolysin sequences, phages exhibited significant differences in host range. Detailed analysis unveiled diverse receptor-binding protein sequences, with similar structures but distinct ligand-binding sites. These findings emphasize the importance of ligand-binding sites of receptor-binding proteins. Additionally, bacterial reduction curve and virulence index revealed that Enteritidis phages inhibit bacterial growth even at low concentrations, unlike Infantis and Kentucky phages.