Binding Sites Research Articles

Synthesis and biological assessment of novel 4H-chromene-3-carbonitrile derivatives as tyrosinase inhibitors.

Excessive activity of the tyrosinase enzyme during melanogenesis results in hyperpigmentation in the skin. To address this issue, there is a need to develop effective tyrosinase inhibitors as a treatment for hyperpigmentation. In this study, we synthesized some novel 4H-chromene-3-carbonitrile compounds (6a-o) and assessed their inhibitory activities against tyrosinase, comparing them with kojic acid, which is known as a positive control. Compound 6f emerged as the most effective inhibitor, with an IC50 of 35.38 ± 2.12 µM. Kinetic studies of 6f exhibited competitive inhibition, with Ki = 16.15 µM. Molecular docking studies highlighted the importance of π-π stacking and hydrogen bonding interactions within the binding site. Molecular dynamics simulations showed that the R-enantiomer 6f exhibited superior binding stability compared to the S-enantiomer, with a lower standard deviation of RMSD and more persistent interactions with the key active site residues. These findings underscore the potential of the R-enantiomer of compound 6f as a potent tyrosinase inhibitor and provide insights for developing effective treatments for hyperpigmentation and related skin conditions.

Development of a FUT8 Inhibitor with Cellular Inhibitory Properties.

Core fucosylation is catalyzed by α-1,6-fucosyltransferase (FUT8), which fucosylates the innermost GlcNAc of N-glycans. Given the association of FUT8 with various diseases including cancer, selective FUT8 inhibitors applicable to in vivo or cell-based systems are highly sought-after. Here, we report the discovery of a compound that selectively inhibits FUT8 in cell-based assays. High-throughput screening revealed a FUT8-inhibiting pharmacophore, and further structural optimization yielded an inhibitor with a KD of 49 nM. Notably, this binding occurs only in the presence of GDP (a product of the enzymatic reaction catalyzed by FUT8). Mechanistic studies suggested that this inhibitor generates a highly reactive naphthoquinone methide derivative at the binding site in FUT8, which subsequently reacts with FUT8. Furthermore, prodrug derivatization of this inhibitor improved its stability, enabling suppression of core fucose expression and subsequent EGFR and T-cell signaling in cell-based assays, paving the way for the development of drugs targeting core fucosylation.

Nucleic acid binding affinity and antioxidant activity of N-m-Tolyl-4-Chlorophenoxyacetohydroxamicacid.

Hydroxamic acids represent a group of weak organic acids, both naturally occurring and synthetically derived, characterized by the general formula RC(= O)N(R'OH). In this study, we investigated the binding behavior of N-m-tolyl-4-chlorophenoxyaceto hydroxamic acid with calf thymus DNA (ct-DNA) and torula yeast RNA (t-RNA) through a combination of techniques including UV-visible spectroscopy, fluorescence emission analysis, viscometry, and computational simulations using AutoDock4 software. Our findings reveal that the mode of binding between the compound and the nucleic acids is consistent with intercalation. Competitive binding experiments demonstrated that the complex competes effectively with ethidium bromide (EB) for binding to ct-DNA/t-RNA, displacing EB from its binding sites. Additionally, the introduction of the compound into the DNA-EB system resulted in a quenching of fluorescence emission peaks. Analysis of absorption spectra indicated a red shift and hypochromic shift when the compound interacted with DNA, further supporting the intercalative binding mode. The calculated binding constant (Kb) value for the compound is 6.62 × 104M-1 and 5.40 × 103M-1 indicating a strong interaction with ct-DNA and t-RNA respectively. We determined the Stern-Volmer constants for ct-DNA and t-RNA as 9.96 × 104M-1 and 8.13 × 105M-1, respectively. The binding free energy values for ct-DNA/t-RNA were calculated to be -3.741 × 107 and -5.425 × 108kcal/mol, respectively. Viscometric studies corroborated the UV results, showing a continuous increase in relative viscosity of ct-DNA/t-RNA solutions with the addition of the optimal hydroxamic acid concentration. Furthermore, we assessed the antioxidant activity of the compound using DPPH-radical scavenging and β-carotene linoleic acid assays. Gel electrophoresis results demonstrated the compound's remarkable efficacy in preventing DNA damage. Collectively, all experimental evidence supports the conclusion that N-m-tolyl-4-chlorophenoxyaceto hydroxamic acid binds to ct-DNA/t-RNA through an intercalative mechanism, which is consistent with our molecular docking simulations.

Modeling Of the Structure of the tRolC Protein of Nicotiana tabacum and Its Functional Relation to Other Proteins

The trolC gene is a homologue of the rolC oncogene of Agrobacterium rhizogenes and most likely got into Nicotiana tabacum genome during horizontal gene transfer. This gene is quite conserved and is expressed in young organs, but its functions, like the agrobacterium rolC gene, remain unknown. The putative structure of the tRolC protein of Nicotiana tabacum, predicted using the I-TASSER service, was analyzed in the study. The presence of amino acid residues in the tRolC protein of the active center and binding site, as well as a protein-binding and ARTT loop, similar in structure to protein 6b of Agrobacterium vitis, indicates that they perform similar biological functions. Based on the data, a hypothesis was put forward about the presence of ADP-ribosyltransferase activity in the tRolC protein, which allows it to influence the growth and stress tolerance, as well as the biosynthesis of secondary metabolites by influencing the regulation of gene expression through interaction with transcription factors, RNA interference, or the manifestation of histone-like activity.

Cooperativity among clustered κB sites within promoters and enhancers dictates transcriptional specificity of NF-κB-RelA along with specific cofactors.

The functional role of weak DNA binding sites for transcription factor (TF) recruitment and gene expression remains largely unknown. Our study reveals that the weak NF-κB DNA binding sites, which are abundant in gene promoters and enhancers, appear in clusters and exhibit minimal to undetectable NF-κB binding activity in isolation in vitro, yet they play prominent roles in gene regulation within native context in cells. We found nuclear concentration of RelA/p65, the predominant NF-κB, is approximately 0.2 µM in stimulated cells, challenging the idea that these weak κB sites operate through mass action-dependent binding mechanisms. Through proteomic analysis, we identified a range of nuclear factors, including various other TFs, interacting with RelA at these κB-sites. ChIP-seq, RNA-seq and phase-separated condensation analyses suggest these additional TFs, referred to as the cofactors of NF-κB, facilitate dynamic recruitment of NF-κB to clustered κB sites of specific target genes. Overall, our findings demonstrate the collective contribution of both strong and weak κB sites in occupancy of NF-κB at the promoters and enhancers, with the recruitment facilitated by a variety of cofactors. This congregation of multiple factors forming larger dynamic complexes appearing as a transcription condensate is likely to be common to all transcriptional programs.

Potent and Selective SETDB1 Covalent Negative Allosteric Modulator Reduces Methyltransferase Activity in Cells.

A promising drug target, SETDB1, is a dual Kme reader and methyltransferase, which has been implicated in cancer and neurodegenerative disease progression. To help understand the role of the triple Tudor domain (3TD) of SETDB1, its Kme reader, we first identified a low micromolar small molecule ligand, UNC6535, which occupies simultaneously both the TD2 and TD3 reader binding sites. Further optimization led to the discovery of UNC10013, the first covalent 3TD ligand targeting Cys385 of SETDB1. UNC10013 is potent with a k inact /K I of 1.0 x 10 6 M -1 s -1 and demonstrated proteome-wide selectivity. In cells, negative allosteric modulation of SETDB1-mediated Akt methylation was observed after treatment with UNC10013. Therefore, UNC10013 is a potent, selective and cell-active covalent ligand for the 3TD of SETDB1, demonstrating negative allosteric modulator properties and making it a promising tool to study the biological role of SETDB1 in disease progression.

Fine Mapping Regulatory Variants by Characterizing Native CpG Methylation with Nanopore Long-Read Sequencing.

5-methylcytosine (5mC) is the most common chemical modification occurring on the CpG sites across the human genome. Bisulfite conversion combined with short-read whole genome sequencing can capture and quantify the modification at single nucleotide resolution. However, the PCR amplification process could lead to duplicative methylation patterns and introduce 5mC detection bias. Additionally, the limited read length also restricts co-methylation analysis between distant CpG sites. The bisulfite conversion process presents a significant challenge for detecting variant-specific methylation due to the destruction of allele information in the sequencing reads. To address these issues, we sought to characterize the human methylation profiling with the nanopore long-read sequencing, aiming to demonstrate its potential for long-range co-methylation analysis with native modification call and intact allele information retained. In this regard, we first analyzed the nanopore demo data in the adaptive sampling sequencing run targeting all human CpG islands. We applied the linkage disequilibrium (LD) R2 to calculate the co-methylation in nanopore data, and further identified 27,875, 50,481, 26,542 and 51,189 methylation haplotype blocks (MHB) in COLO829, COLO829BL, HCC1395 and HCC1395BL cell lines, respectively. Interestingly, while we found that majority of the co-methylation were in a short range (≤200bp), a small portion (1~3%) showed long distance (≥1,000bp), suggesting potential remote regulatory mechanisms across the genome. To further characterize the epigenetic changes related to transcription factor binding, we profiled the 5mC percentage changes surrounding various motif sites in JASPAR collection and found that CTCF and KLF5 binding sites showed reduced methylation, while FOXE1 and ZNF354A sites showed increased methylation. To further investigate the allele-specific 5mCG in the prostate genome, we designed a target region covering methylation quantitative trait loci (mQTL) and genome-wide association study (GWAS) risk germline variants and generated long reads with adaptive sampling run in the 22Rv1 cell line. To identify the allele-specific methylation in the 22Rv1 cell line, we performed long-read based phasing and compared the 5mCG signals between the two haplotypes. As a result, we identified 6,390 haplotype-specific methylated regions in the 22Rv1 cell line (p-MWU ≤ 1e-5 and delta ≥ 50%). By examining haplotype-specific methylated regions near the phasing variants, we identified examples of allele-specific methylated regions that showed allelespecific accessibility in the ATAC-seq data. By further integrating the ATAC-seq data of 22Rv1, we found that methylation levels were negatively correlated with chromatin accessibility at the genome-wide scale. Our study has revealed native methylome profiling while preserving haplotype information, offering a novel approach to uncovering the regulatory mechanisms of the human prostate genome.

Regulatory mechanism of C4-dicarboxylates in cyclo (Phe-Pro) production

Cyclo (Phe-Pro) (cFP), a cyclic dipeptide with notable antifungal, antibacterial, and antiviral properties, shows great promise for biological control of plant diseases. Produced as a byproduct by non-ribosomal peptide synthetases (NRPS), the regulatory mechanism of cFP biosynthesis remains unclear. In a screening test of 997 Tn5 mutants of Burkholderia seminalis strain R456, we identified eight mutants with enhanced antagonistic effects against Fusarium graminearum (Fg). Among these, mutant 88’s culture filtrate contained cFP, confirmed through HPLC and LC-MS, which actively inhibited Fg. The gene disrupted in mutant 88 is part of the Dct transport system (Dct-A, -B, -D), responsible for C4-dicarboxylate transport. Knockout mutants of Dct genes exhibited higher cFP levels than the wild type, whereas complementary strains showed no significant difference. Additionally, the presence of exogenous C4-dicarboxylates reduced cFP production in wild type R456, indicating that these substrates negatively regulate cFP synthesis. Given that cFP synthesis is related to NRPS, we previously identified an NRPS cluster in R456, horizontally transferred from algae. Specifically, knocking out gene 2061 within this NRPS cluster significantly reduced cFP production. A Fur box binding site was predicted upstream of gene 2061, and yeast one-hybrid assays confirmed Fur protein binding, which increased with additional C4-dicarboxylates. Knockout of the Fur gene led to up-regulation of gene 2061 and increased cFP production, suggesting that C4-dicarboxylates suppress cFP synthesis by enhancing Fur-mediated repression of gene 2061.

Molecularly Imprinted Polymers Electrochemical Sensing: The Effect of Inhomogeneous Binding Sites on the Measurements. A Comparison between Imprinted Polyaniline versus nanoMIP-Doped Polyaniline Electrodes for the EIS Detection of 17β-Estradiol.

Molecularly imprinted polymers (MIPs) are synthetic receptors made by template-assisted synthesis. MIPs might be ideal receptors for sensing devices, given the possibility to custom-design selectivity and affinity toward a targeted analyte and their robustness and ability to withstand harsh conditions. However, the synthesis of MIP is an inherently random process that produces a statistical distribution of binding sites, characterized by a variety of affinities. This is verified both for bulk MIP materials and for MIP's thin layers. In the present work, we aimed at assessing the effects of inhomogeneous versus homogeneous imprinted binding sites on electrochemical sensing measurements, and the possible implications on the sensor's performance. In the example of an Electrochemical Impedance Spectroscopy (EIS) sensor for the 17β-estradiol (E2) hormone, the scenario of inhomogeneous binding sites was studied by modifying electrodes with an E2-MIP polyaniline (PANI) thin layer, called the "Imprinted PANI layer". In contrast, the condition of discrete and uniform binding sites was epitomized by electrodes modified with a thin PANI layer purposedly doped with E2-MIP nanoparticles (nanoMIPs), which were referred to as "nanoMIP-doped PANI". The behaviors of the two EIS sensors were compared. Interestingly, the sensitivity of the nanoMIP-doped PANI was almost twice with respect to that of the imprinted PANI layer, strongly suggesting that the homogeneity of the binding sites has a fundamental role in the sensor's development. The nanoMIP-doped PANI sensor, which showed a response for E2 in the range 36.7 pM-36.7 nM and had a limit of detection of 2.86 pg/mL, was used to determine E2 in wastewater.

The impact of host microRNAs on the development of conserved mutations of SARS-CoV-2.

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has undergone various genetic alterations due to evolutionary pressures exerted by host cells, including intracellular antiviral mechanisms such as targeting by human microRNAs (miRNAs). This study investigates the impact of miRNAs hsa-miR-3132 and hsa-miR-4650 on the viral genome. Sequence alignment revealed conserved mutations in the binding sites of these miRNAs in adapted strains compared to the original Wuhan-Hu-1 strain, leading to their deletion. Despite modest expression of these miRNAs in SARS-CoV-2 target tissues, their efficacy against mutant strains is reduced due to the loss of binding sites. Structural analysis indicates that the mutant genome is more stable than the Wuhan-Hu-1 genome. Luciferase and virus titration assays demonstrate that hsa-miR-3132 and hsa-miR-4650 effectively target the Nsp3 gene in the Wuhan-Hu-1 strain but not in mutant strains lacking their binding sites. These findings suggest that the observed mutations help the virus evade selective pressure from human miRNAs, contributing to its adaptation.

Analysis of chicken LGALSL (galectin-related protein) gene’s proximal promoter and its control by Krüppel-like factors 3 and 7

The Galectin-Related Protein (GRP), encoded by the LGALSL gene, assigned to the protein family of β-galactoside-binding Galectins, has lost carbohydrate-binding abilities. Its chicken homolog (C-GRP) occurs in the bursa of Fabricius’ epithelial and B cells. Our study investigates the unknown regulatory mechanisms controlling its expression by analyzing the promoter region of the chicken (C-)LGALSL gene in chicken cells. We aimed to identify the sequence elements of the C-LGALSL gene promoter responsible for maximum activity and transcription factors (TFs) that can modulate this activity. Using luciferase reporter assays, we investigated deletion variants of the 5′ region (−2480 bp to +26 bp). Through in silico analyses and site-directed mutagenesis, we explored potential transcription factor binding sites, identified crucial transcription factors through transient overexpression and tested its direct binding by ChIP. Our findings highlight that the region from −274 to −75 bp, conserved among bird species, is crucial for promoter regulation. Among other tested factors, only the chicken (ch) Krüppel-like factors, chKLF3 and chKLF7, modulate the promoter’s activity. The TFs chKLF3 acts as a repressor, and chKLF7 as an activator, although direct binding could not be confirmed. In conclusion, chKLF3 and chKLF7 contribute, in contrast to other factors with binding sites in the region from −274 to −75 bp, to C-LGALSL gene promoter regulation with a balanced impact on activity.

An in silico design method of a peptide bioreceptor for cortisol using molecular modelling techniques

Cortisol is established as a reliable biomarker for stress prompting intensified research in developing wearable sensors to detect it via eccrine sweat. Since cortisol is present in sweat in trace quantities, typically 8–140 ng/mL, developing such biosensors necessitates the design of bioreceptors with appropriate sensitivity and selectivity. In this work, we present a systematic biomimetic methodology and a semi-automated high-throughput screening tool which enables rapid selection of bioreceptors as compared to ab initio design of peptides via computational peptidology. Candidate proteins from databases are selected via molecular docking and ranked according to their binding affinities by conducting automated AutoDock Vina scoring simulations. These candidate proteins are then validated via full atomistic steered molecular dynamics computations including umbrella sampling to estimate the potential of mean force using GROMACS version 2022.6. These explicit molecular dynamic calculations are carried out in an eccrine sweat environment taking into consideration the protein dynamics and solvent effects. Subsequently, we present a candidate baseline peptide bioreceptor selected as a contiguous sequence of amino acids from the selected protein binding pocket favourably interacting with the target ligand (i.e., cortisol) from the active binding site of the proteins and maintaining its tertiary structure. A unique cysteine residue introduced at the N-terminus allows orientation-specific surface immobilization of the peptide onto the gold electrodes and to ensure exposure of the binding site. Comparative binding affinity simulations of this peptide with the target ligand along with commonly interfering species e.g., progesterone, testosterone and glucose are also presented to demonstrate the validity of this proposed peptide as a candidate baseline bioreceptor for future cortisol biosensor development.

Bacterial amino acid chemotaxis: a widespread strategy with multiple physiological and ecological roles.

Chemotaxis is the directed, flagellum-based movement of bacteria in chemoeffector gradients. Bacteria respond chemotactically to a wide range of chemoeffectors, including amino, organic, and fatty acids, sugars, polyamines, quaternary amines, purines, pyrimidines, aromatic hydrocarbons, oxygen, inorganic ions, or polysaccharides. Most frequent are chemotactic responses to amino acids (AAs), which were observed in numerous bacteria regardless of their phylogeny and lifestyle. Mostly chemoattraction responses are observed, although a number of bacteria are repelled from certain AAs. Chemoattraction is associated with the important metabolic value of AAs as growth substrates or building blocks of proteins. However, additional studies revealed that AAs are also sensed as environmental cues. Many chemoreceptors are specific for AAs, and signaling is typically initiated by direct ligand binding to their four-helix bundle or dCache ligand-binding domains. Frequently, bacteria possess multiple AA-responsive chemoreceptors that at times possess complementary AA ligand spectra. The identification of sequence motifs in the binding sites at dCache_1 domains has permitted to define an AA-specific family of dCache_1AA chemoreceptors. In addition, AAs are among the ligands recognized by broad ligand range chemoreceptors, and evidence was obtained for chemoreceptor activation by the binding of AA-loaded solute-binding proteins. The biological significance of AA chemotaxis is very ample including in biofilm formation, root and seed colonization by beneficial bacteria, plant entry of phytopathogens, colonization of the intestine, or different virulence-related features in human/animal pathogens. This review provides insights that may be helpful for the study of AA chemotaxis in other uncharacterized bacteria.

Mechanism of Dual-Site Recognition in a Classic DNA Aptamer.

Nucleic acid aptamers possess unique advantages in specific recognition. However, the lack of in-depth investigation into their dynamic recognition mechanisms has restricted their rational design and potential applications in fields such as biosensing and targeted therapy. We herein utilized enhanced sampling molecular dynamics to address affinities of adenosine monophosphate (AMP) to the dual binding sites in the DNA aptamer, focusing on the dynamic recognition mechanism and pathways. The present results indicate that in addition to the widely known intermolecular interactions, inequivalence of chemical environments of the two binding sites leads to slightly higher stability of AMP binding to the site proximal to the aptamer terminus. In the presence of two AMPs captured by the two sites, each binding free energy is enhanced. In particular, an additional hydrogen bond of AMP to A10 is introduced in the dual-site binding complex, which increases the binding energy from -4.25 ± 0.47 to -9.48 ± 0.33 kcal mol-1 in the site close to the loop. For the dual-site recognition process, the free energy landscape and minimum free energy pathway calculations elucidate the crucial role of electrostatic interactions between the AMP phosphate groups and Na+ ions in positively cooperative binding mechanisms.

Expansion and diversification of the Glycine max (Gm) ERD15-like subfamily of the PAM2-like superfamily.

Despite modulating senescence and drought responses, the GmERD15-like subfamily members are differentially induced by multiple stresses and diverge partially in stress signaling functions. The PAM2 motif represents a binding site for poly (A)-binding proteins (PABPs), often associated with RNA metabolism regulation. The PAM2-containing protein ERD15 stands out as a critical regulator of diverse stress responses in plants. Despite the relevance of the PAM2 motif, a comprehensive analysis of the PAM2 superfamily and ERD15-like subfamily in the plant kingdom is lacking. Here, we provide an extensive in silico analysis of the PAM2 superfamily and the ERD15-like subfamily in soybean, using Arabidopsis and rice sequences as prototypes. The Glycine max ERD15-like subfamily members were clustered in pairs, likely originating from DNA-based gene duplication, as the paralogs display high sequence conservation, similar exon/intron genome organization, and are undergoing purifying selection. Complementation analyses of an aterd15 mutant demonstrated that the plant ERD15-like subfamily members are functionally redundant in response to drought, osmotic stress, and dark-induced senescence. Nevertheless, the soybean members displayed differential expression profiles, biochemical activity, and subcellular localization, consistent with functional diversification. The expression profiles of Glyma04G138600 under salicylic acid (SA) and abscisic acid (ABA) treatments differed oppositely from those of the other GmERD15-like genes. Abiotic stress-induced coexpression analysis with soybean PABPs showed that Glyma04G138600 was clustered separately from other GmERD15s. In contrast to the AtERD15 stress-induced nuclear redistribution, Glyma04G138600 and Glyma02G260800 localized to the cytoplasm, while Glyma03G131900 fractionated between the cytoplasm and nucleus under normal and stress conditions. These data collectively indicate that despite modulating senescence and drought responses, the GmERD15-like subfamily members are differentially induced by multiple stresses and may diverge partially in stress signaling functions.

The combined effects of bisphenol S and hexavalent chromium on alpha-glucosidase: Intermolecular interaction, structural and functional changes

The co-contamination of heavy metal ions and organic pollutants has posed a threat to human health. Herein, this study investigated the intermolecular interactions of bisphenol S (BPS) and hexavalent chromium (Cr(VI)) under both individual and coexisting conditions, with alpha-glucosidase (AG), a key enzyme in carbohydrate metabolism, and the corresponding effects on the structure and function of AG. Multiple spectroscopic and molecular docking methods were employed to conduct the investigation in vitro and in silico. The results indicated that both BPS and Cr(VI) quenched the fluorescence of AG via a combined static and dynamic quenching processes. At 310 K, the binding constants of AG with BPS in the AG-BPS and (AG-Cr(VI))-BPS systems were 1.84 × 104 and 2.03 × 104 L mol−1, and the binding constants of AG with Cr(VI) in the AG-Cr(VI) and (AG-BPS)-Cr(VI) systems were 6.14 × 103 and 4.35 × 103 L mol−1. Cr(VI) could significantly affect the binding site of BPS in AG, while BPS had a minimal impact on the binding site of Cr(VI) in AG. BPS and Cr(VI) caused varied structural alterations of AG, and the impact of their coexistence on the structure of AG was related to the order in which they were added. Both BPS and Cr(VI) had a concentration-related effect on AG activity. This study provides valuable insights into the molecular mechanisms underlying the combined toxic effects of BPS and Cr(VI) on AG, highlighting the potential health risks associated with their environmental co-exposure.

Theoretical investigation for the proline effect on peptide fragmentation induced by matrix-assisted laser desorption/ionization in-source decay with an oxidizing matrix

The use of an oxidizing matrix for matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) specifically induces the cleavage of Cα-C bonds on the peptide backbone, except for bonds located on the N-terminal side of proline residues. To examine the effect of Pro residues on bond cleavage induced by MALDI-ISD with an oxidizing matrix, the small dipeptides AcAA-NH2 and AcAP-NH2 were used as models. As the fragmentation is initiated by electron transfer from the peptide to the oxidizing matrix, the dissociation chemistry of the cation radical forms of the model peptides [AcAA-NH2]+• and [AcAP-NH2]+• was investigated using quantum chemistry calculations. The [AcAA-NH2]+• can produce fragment ions not only due to Cα–C bond cleavage, but also peptide bond cleavage. Quantum chemistry calculations indicated that peptide bond cleavage of [AcAA-NH2]+• occurs more slowly compared to Cα–C bond cleavage. Instead of Cα–C bond cleavage, the bond on the N-terminal side of Pro residue undergoes peptide bond cleavage during MALDI-ISD with an oxidizing matrix, due to the lack of an amide hydrogen in the Pro residue. The [AcAP-NH2]+• undergoes proton migration from the δ-carbon of the Pro residue. Depending on the proton binding site in the peptide cation radical, the peptide bond cleavage of [AcAP-NH2]+• results in the formation of either [b1]+, and [y1]•, or [a1]• and [y1]+. These theoretical results are consistent with experimental findings, and the newly proposed mechanism involving peptide cation radical formation followed by proton migration provides a more accurate model for the MALDI-ISD processes.

Environmentally friendly molecularly imprinted polymers as an insert for SPE type columns in the gentamicin monitoring process

The quantity and variety of micro-pollutants infiltrating water resources have increased rapidly in recent times. The appearance of many harmful substances in the waters has resulted in so-called chemical cocktails which significantly contribute to the deterioration of water quality. Additionally, the variety of these compounds, often similar to each other in terms of molecular weights, makes their separation and identification very difficult. In this paper we present the possibility of using self-regenerating mechanism of molecularly imprinted polymers to measure the concentration of micropollutants in the aquatic environment. Molecularly imprinted polymers toward gentamicin were prepared by monomer polymerization in aqueous solution at ambient temperature. Results from computer-based molecular modelling demonstrated potential binding sites between gentamicin and functional monomers in water. Various compositions of polymerization mixtures were tested. The ratio of monomers to each other was 1.1:1.4:0.0015 and 1:1:1 for N-isopropylacrylamine:acrylamide:acrylic acid, respectively. For each composition, various amounts of the standard were tested: 0, 3, 5, 7, 10,15 mol% in relation to monomers. The best results were obtained for 5 % gentamicin with an excess of acrylamide in relation to the other monomers. Sorption for this system was 0.783 mg/g at ambient temperature and desorption 0.593 at 4 °C. The synthesized materials, thanks to the incorporation of thermosensitive poly(N-isopropylacrylamide) into their structures, were able to release 89 % of adsorbed gentamicin. This made it possible to use the designed SPE columns repeatably with similar efficiency. The prepared materials were selective in the presence of other antibiotics like amoxicillin and norfloxacin.

Dynamics of the Pre-Powerstroke Myosin Lever Arm and the Effects of Omecamtiv Mecarbil.

The binding of small molecules to sarcomeric myosin can elicit powerful effects on the chemomechanical cycle, making them effective therapeutics in the clinic and research tools at the benchtop. However, these myotropes can have complex effects that act on different phases of the crossbridge cycle and which depend on structural, dynamic, and environmental variables. While small molecule binding sites have been identified crystallographically and their effects on contraction studied extensively, small molecule-induced dynamic changes that link structure-function are less studied. Here, we use molecular dynamics simulations to explore how omecamtiv mecarbil (OM), a cardiac myosin-specific myotrope, alters the coordinated dynamics of the lever arm and the motor domain in the pre-powerstroke state. We show that the lever arm adopts a range of orientations and find that different lever arm orientations are accompanied by changes in the hydrogen bonding patterns near the converter. We find that the binding of OM to myosin reduces the conformational heterogeneity of the lever arm orientation and also adjusts the average lever arm orientation. Finally, we map out the distinct conformations and ligand-protein interactions adopted by OM. These results uncover some structural factors that govern the motor domain-tail orientations and the mechanisms by which OM primes the pre-powerstroke myosin heads.

Unveiling Temperature-Induced Structural Phase Transformations and CO2 Binding Sites in CALF-20.

The increase in atmospheric carbon dioxide concentration linked to climate change has created a need for new sorbents capable of separating CO2 from exhaust gases. Recently, an easily produced metal-organic framework, CALF-20, was shown to withstand over 450,000 adsorption/desorption cycles in steam and wet acid gases. Further development and industrial application of such materials require an understanding of the observed processes. Herein, we demonstrate that conditioning as-synthesized CALF-20 single crystal transforms it into a different phase, γ-CALF-20. The transformation resulted in significant structural changes, including the binding of water molecules to Zn(II), accompanied by a reduction of 9% in the unit cell volume. Our experimental findings were supported by the energy-volume dependence of CALF-20 in the presence and absence of water molecules calculated from density functional theory. We have also monitored the sorption process of the dominant greenhouse gas, CO2, on the initial phase of CALF-20 at atomic resolution using in situ single-crystal X-ray diffraction under specific pressure. The new understanding of CALF-20 chemistry from these studies should facilitate development of novel sorbents for gas adsorption technologies.