kDa Domain Research Articles

The enzymatic properties of Arabidopsis thaliana DNA polymerase λ suggest a role in base excision repair

Base excision repair (BER) generates gapped DNA intermediates containing a 5′-terminal 2-deoxyribose-5-phosphate (5′-dRP) group. In mammalian cells, gap filling and dRP removal are catalyzed by Pol β, which belongs to the X family of DNA polymerases. In higher plants, the only member of the X family of DNA polymerases is Pol λ. Although it is generally believed that plant Pol λ participates in BER, there is limited experimental evidence for this hypothesis. Here we have characterized the biochemical properties of Arabidopsis thaliana Pol λ (AtPol λ) in a BER context, using a variety of DNA repair intermediates. We have found that AtPol λ performs gap filling inserting the correct nucleotide, and that the rate of nucleotide incorporation is higher in substrates containing a C in the template strand. Gap filling catalyzed by AtPol λ is most efficient with a phosphate at the 5′-end of the gap and is not inhibited by the presence of a 5′-dRP mimic. We also show that AtPol λ possesses an intrinsic dRP lyase activity that is reduced by mutations at two lysine residues in its 8-kDa domain, one of which is present in Pol λ exclusively and not in any Pol β homolog. Importantly, we also found that the dRP lyase activity of AtPol λ allows efficient completion of uracil repair in a reconstituted short-patch BER reaction. These results suggest that AtPol λ plays an important role in plant BER.

Fibronectin on target cells attenuates natural cytotoxicity of NK cells via myeloid immune checkpoint ILT3/LILRB4/gp49B.

Natural killer (NK) cells play pivotal roles in innate immunity as well as in anti-tumor responses via natural killing, while their activity is tightly regulated by cell-surface inhibitory receptors. Immunoglobulin-like transcript 3/leukocyte immunoglobulin-like receptor B4 (ILT3/LILRB4, also known as gp49B in mice) is an inhibitory receptor expressed on activated NK cells as well as myeloid-lineage cells. The common physiologic ligand of human LILRB4 and gp49B was identified very recently as fibronectin, particularly the N-terminal 30 kDa domain (FN30). We hypothesized that LILRB4 could bind fibronectin on target cells in trans together with integrins, classical fibronectin receptors, in cis and deliver an inhibitory signal in NK cells, leading to attenuated natural killing. Flow cytometric and confocal microscopic analyses of NK cell-surface gp49B and integrins suggested that these novel and classical fibronectin receptors, respectively, co-engage fibronectin immobilized on a culture plate. Biochemical analyses indicated that tyrosine phosphorylation of spleen tyrosine kinase was augmented in gp49B-deficient NK cells upon binding to the immobilized fibronectin. While surface fibronectin-poor YAC-1 cells were evenly sensitive as to natural killing of both gp49B-positive and -negative NK cells, the killing of fibronectin-rich Lewis lung carcinoma cells, but not the FN30-knockout cells, was augmented among gp49B-deficient NK cells. These results suggest that the natural cytotoxicity of NK cells is negatively regulated through LILRB4/gp49B sensing fibronectin on target cells, which sheds light on the unexpected role of LILRB4 and fibronectin as a potential attenuator of NK cell cytotoxicity in the tumor microenvironment.

Dilated cardiomyopathy mutation (E525K) in human beta-cardiac myosin enhances actin-activated phosphate release but stabilizes the auto-inhibited super-relaxed state.

Mutations in beta-cardiac myosin (M2β) are a common cause of inherited cardiomyopathies including dilated (DCM) and hypertrophic cardiomyopathy (HCM). We investigated the impact of a DCM mutation (E525K) in the human M2β motor domain, which has been shown to stabilize both the interacting heads motif (IHM) and auto-inhibited super-relaxed (SRX) state via head-tail electrostatic interactions in 15 heptad heavy meromyosin (M2β HMM). These findings suggest E525K may reduce muscle force and power by triggering an increase in the IHM/SRX state. However, it is unclear how E525K impacts the intrinsic motor properties of M2β. Thus, we introduced the mutation into single-headed human cardiac myosin subfragment 1 (M2β-S1), unable to form the IHM, and examined its impact on the ATPase mechanism. We revealed that E525K induces a 2.8-fold increase in maximum steady-state actin-activated ATPase activity (kcat), a 6-fold decrease in the actin concentration at which ATPase is half maximal (KATPase), and a 3.4-fold increase in the fast phase of actin-activated phosphate release. Glutamate 525 is located in the conserved activation loop that is proposed to trigger phosphate release by interacting with the negatively charged N-terminus of actin. We propose the mutation may enhance the rate of rotation of the lower 50-kDa domain, movement of the relay helix/switch II, and lever arm rotation associated with the power stroke. Thus, we will directly examine the impact on the actin-activated power stroke utilizing a FRET approach that measures lever arm rotation. Our results suggest the E525K mutation triggers an increase in intrinsic motor properties, while its ability to stabilize the SRX/IHM state in the myosin dimer likely dominates the molecular mechanism that leads to decreased muscle force and power and DCM pathogenesis.

A synergistic investigation of azo-thiazole derivatives incorporating thiazole moieties: a comprehensive exploration of their synthesis, characterization, computational insights, solvatochromism, and multimodal biological activity assessment.

In the present study, a novel series of azo-thiazole derivatives (3a-c) containing a thiazole moiety was successfully synthesized. The structure of these derivatives was examined by spectroscopic techniques, including 1H NMR, 13C NMR, FT-IR, and HRMS. Further, the novel synthesized compounds were evaluated for their in vitro biological activities, such as antibacterial and anti-inflammatory activities, and an in silico study was performed. The antibacterial results demonstrated that compounds 3a and 3c (MIC = 10 μg mL-1) have a notable potency against Staphylococcus aureus compared to azithromycin (MIC = 40 μg mL-1). Alternatively, compound 3b displayed a four-fold higher potency (24 recovery days, 1.83 mg day-1) than Hamazine (28 recovery days, 4.14 mg day-1) in promoting burn wound healing, and it also exhibited a comparable inhibitory activity against screened bacterial pathogens compared to the reference drug. Docking on 1KZN, considering the excellent impact of compounds on the crystal structure of E. coli1KZN, a 24 kDa domain, in complex with clorobiocin, indicated the close binding of compounds 3a-c with the active site of the 1KZN protein, which is consistent with their observed biological activity. Additionally, we conducted molecular dynamics simulations on the docked complexes of compounds 3a-c with 1KZN retrieved from the PDB to assess their stability and molecular interactions. Furthermore, we assessed their electrochemical characteristics via DFT calculations. Employing PASS and pkCSM platforms, we gained insights into controlling the bioactivity and physicochemical features of these compounds, highlighting their potential as new active agents.

Myeloid immune checkpoint ILT3/LILRB4/gp49B can co-tether fibronectin with integrin on macrophages.

LILRB4 (B4, also known as ILT3/CD85k) is an immune checkpoint of myeloid lineage cells, albeit its mode of function remains obscure. Our recent identification of a common ligand for both human B4 and its murine ortholog gp49B as the fibronectin (FN) N-terminal 30 kDa domain poses the question of how B4/gp49B regulate cellular activity upon recognition of FN in the plasma and/or the extracellular matrix. Since FN in the extracellular matrix is tethered by FN-binding integrins, we hypothesized that B4/gp49B would tether FN in cooperation with integrins on the cell surface, thus they should be in close vicinity to integrins spatially. This scenario suggests a mode of function of B4/gp49B by which the FN-induced signal is regulated. The FN pull-down complex was found to contain gp49B and integrin β 1 in bone marrow-derived macrophages. The confocal fluorescent signals of the three molecules on the intrinsically FN-tethering macrophages were correlated to each other. When FN-poor macrophages adhered to culture plates, the gp49-integrin β 1 signal correlation increased at the focal adhesion, supporting the notion that gp49B and integrin β 1 become spatially closer to each other there. Adherence of RAW264.7 and THP-1 cells to immobilized FN induced phosphorylation of spleen tyrosine kinase, whose level was augmented under B4/gp49B deficiency. Thus, we concluded that B4/gp49B can co-tether FN in cooperation with integrin in the cis configuration on the same cell, forming a B4/gp49B-FN-integrin triplet as a regulatory unit of a focal adhesion-dependent pro-inflammatory signal in macrophages.

Characterization of proteolytic degradation products of vaginally administered bovine lactoferrin.

When bovine lactoferrin (bLF) contacts human vaginal fluid (VF) it is subjected to proteolytic degradation. This report describes fragmentation patterns of bLF dosed vaginally in clinical trials or incubated ex vivo with VF. A consensus pattern of fragments was observed in samples from different women. The 80 kDa bLF molecule is initially cleaved between its homologous 40 kDa domains, the N-lobe and C-lobe, and then degraded into sub-fragments and mixtures of small peptides. We characterized this fragmentation process by polyacrylamide gel electrophoresis, western blotting, chromatographic separation, and mass spectral sequence analysis. Common to most VF fragmentation patterns were large amounts of an N-lobe 37 kDa fragment and a C-lobe 43 kDa fragment resulting from a single cleavage following tyrosine 324. Both fragments possessed full sets of iron-ligand amino acids and retained iron-binding ability. In some VF samples, alternative forms of large fragments were found, which like the 37+43 kDa pair, totaled 80 kDa. These included 58+22 kDa, 18+62 kDa, and 16+64 kDa forms. In general, the smaller component was from the N-lobe and the larger from the C-lobe. The 18+62 kDa pair was absent in some VF samples but highly abundant in others. This variability suggests multiple endopeptidases are involved, with the 18 kDa fragment’s presence dependent upon the balance of enzymes. Further action of VF endopeptidases produced smaller peptide fragments, and we found evidence that exopeptidases trimmed their N- and C-termini. The 3.1 kDa antimicrobial peptide lactoferricin B was not detected. These studies were facilitated by a novel technique we developed: tricolor western blots, which enabled simultaneous visualization of N- and C-terminal epitopes.

Co-localization of Fibronectin Receptors LILRB4/gp49B and Integrin on Dendritic Cell Surface.

A myeloid immune checkpoint, leukocyte immunoglobulin-like receptor (LILR) B4 (B4, also known as ILT3/CD85k in humans and gp49B in mice) is expressed on dendritic cells (DCs). However, a mode of regulation of DCs by B4/gp49B is not identified yet in relation to the ligand(s) as well as to the counteracting, activation-type receptor. Our recent identification of the physiological/pathological ligand for B4/gp49B as the fibronectin (FN) N-terminal 30-kDa domain poses the question of the relationship between B4/gp49B and a classical FN receptor/cellular activator, integrin, on DCs. Here we showed that FN is not constitutively tethered on the surface of bone marrow-derived cultured DCs (BMDCs) or splenic DCs, even though the FN receptor integrin and gp49B are co-expressed on these cells. Confocal laser scanning microscopic analysis, however, revealed weak correlation of fluorescent signals between gp49B and integrin β1, suggesting their partial co-localization on the BMDC surface even in the absence of FN. We found that the plating of BMDCs onto immobilized FN induced tyrosine phosphorylation of focal adhesion kinase (FAK) and spleen tyrosine kinase (Syk). In the absence of gp49B, while the FAK phosphorylation level was virtually unchanged, that of phosphorylation of Syk was markedly augmented. These results suggested that the immobilized FN induced a crosstalk between gp49B and integrin in terms of the intracellular signaling of BMDCs, in which gp49B suppressed the integrin-mediated pro-inflammatory cascade. Our observations may provide a clue for elucidating the mechanism of the therapeutic efficacy of B4/gp49B blocking in autoimmune disease and cancer.

Molecular basis for inhibition of adhesin-mediated bacterial-host interactions through a peptide-binding domain.

Infections typically begin with pathogens adhering to host cells. For bacteria, this adhesion can occur through specific ligand-binding domains. We identify a 20-kDa peptide-binding domain (PBD) in a 1.5-MDa RTX adhesin of a Gram-negative marine bacterium that colonizes diatoms. The crystal structure of this Ca2+-dependent PBD suggests that it may bind the C termini of host cell-surface proteins. A systematic peptide library analysis reveals an optimal tripeptide sequence with 30-nM affinity for the PBD, and X-ray crystallography details its peptide-protein interactions. Binding of the PBD to the diatom partner of the bacteria can be inhibited or competed away by the peptide, providing a molecular basis for inhibiting bacterium-host interactions. We further show that this PBD is found in other bacteria, including human pathogens such as Vibrio cholerae and Aeromonas veronii. Here, we produce the PBD ortholog from A.veronii and demonstrate, using the same peptide inhibitor, how pathogens may be prevented from adhering to their hosts.

N-Terminally Added Tag Selectively Enhances Heterologous Expression of VacA Cytotoxin Variants from Helicobacter pylori.

Gastric pathogen Helicobacter pylori secretes VacA cytotoxin displaying a high degree of polymorphic variations of which the highest VacA pathogenicity correlates with m1-type variant followed by VacA-m2. To comparatively evaluate expression in Escherichia coli of the mature VacA variants (m1- and m2-types) and their 33- and 55/59-kDa domains fused with His(6) tag at N- or C-terminus. All VacA clones expressed in E. coli TOP10™ were analyzed by SDS-PAGE and Western blotting. VacA inclusions were solubilized under native conditions (~150-rpm shaking at 37°C for 2 h in 20 mM HEPES (pH7.4) and 150 mM NaCl). Membrane-perturbing and cytotoxic activities of solubilized VacA proteins were assessed via liposome-entrapped dye leakage and resazurin- based cell viability assays, respectively. VacA binding to human gastric adenocarcinoma cells was assessed by immunofluorescence microscopy. Side-chain hydrophobicity of VacA was analyzed through modeled structures constructed by homology- and ab initio-based modeling. Both full-length VacA-m1 and 33-kDa domain were efficiently expressed only in the presence of N-terminal extension while its 55-kDa domain was capably expressed with either N- or Cterminal extension. Selectively enhanced expression was also observed for VacA-m2. Protein expression profiles revealed a critical period in IPTG-induced production of the 55-kDa domain with N-terminal extension unlike its C-terminal extension showing relatively stable expression. Both VacA- m1 isolated domains were able to independently bind to cultured gastric cells similar to the full- length toxin, albeit the 33-kDa domain exhibited significantly higher activity of membrane perturbation than others. Membrane-perturbing and cytotoxic activities observed for VacA-m1 appeared to be higher than those of VacA-m2. Homology-based modeling and sequence analysis suggested a potential structural impact of non-polar residues located at the N-terminus of the mature VacA toxin and its 33-kDa domain. Our data provide molecular insights into selective influence of the N-terminally added tag on efficient expression of recombinant VacA variants, signifying biochemical and biological implications of the hydrophobic stretch within the N-terminal domain.

Association Analyses of SNAP25, HNMT, FCHSD1, and DBH Single-Nucleotide Polymorphisms with Parkinson's Disease in a Northern Chinese Population.

PurposeSequencing potentially causal and susceptible genes and genome-wide association studies in samples from Parkinson’s disease (PD) patients has revealed several related loci. The genes for synaptosome-associated protein of 25 kDa (SNAP25), histamine-N-methyltransferase (HNMT), FCH and double SH3 domains 1 (FCHSD1) and dopamine β-hydroxylase (DBH) are candidate loci and have not been studied in a northern Chinese population. We explored the genetic distribution of four single-nucleotide polymorphisms (rs3746544, rs11558538, rs456998, rs129882) located on SNAP25, HNMT, FCHSD1 and DBH, respectively.Patients and MethodsA total of 330 patients with sporadic PD and 332 healthy controls (HCs) were recruited from a northern Chinese population. Polymerase chain reaction restriction fragment length polymorphism was used to genotype these four SNPs.ResultsAfter statistical analyses and correction of the genotyping results, the mutant-allele T in rs456998 of FCHSD1 was found to be significantly related to reducing the PD risk (P = 0.029, OR = 0.754, 95% CI = 0.586–0.971, power = 0.591). However, rs3746544, rs11558538, and rs129882 did not show an association with PD.ConclusionFCHSD1 rs456998 may have a protective role in PD in a northern Chinese population, but more studies are needed to support this suggestion.

ASFV DNA polymerase extends recessed DNAs with catalytic efficiencies outperforming those exerted on gapped DNA substrates

The DNA polymerase from african swine fever virus (ASFV Pol X), lacking both 8 kDa and thumb domains, is the smallest enzyme featuring competence in DNA extension. Here we show that ASFV Pol X features poor filling activity of DNA gaps consisting of 15 bases, and exerts a more efficient action at the expense of DNA substrates containing a recessed end of equal length. We also show that shortening the recessed end of DNA substrates decreases the rate of DNA elongation catalysed by ASFV Pol X. Finally, by means of stopped-flow experiments we were able to determine that DNA binding is a step responsible for restraining the efficiency of ASFV Pol X catalytic action.

Lectin drug conjugate therapy for colorectal cancer.

Drug resistance represents an obstacle in colorectal cancer (CRC) treatment because of its association with poor prognosis. rBC2LCN is a lectin isolated from Burkholderia that binds cell surface glycans that have fucose moieties. Because fucosylation is enhanced in many types of cancers, this lectin could be an efficient drug carrier if CRC cells specifically present such glycans. Therefore, we examined the therapeutic efficacy and toxicity of lectin drug conjugate therapy in CRC mouse xenograft models. The affinity of rBC2LCN for human CRC cell lines HT‐29, LoVo, LS174T, and DLD‐1 was assessed in vitro. The cytocidal efficacy of a lectin drug conjugate, rBC2LCN‐38 kDa domain of pseudomonas exotoxin A (PE38) was evaluated by MTT assay. The therapeutic effects and toxicity for each CRC cell line‐derived mouse xenograft model were compared between the intervention and control groups. LS174T and DLD‐1 cell lines showed a strong affinity for rBC2LCN. In the xenograft model, the tumor volume in the rBC2LCN‐PE38 group was significantly reduced compared with that using control treatment alone. However, the HT‐29 cell line showed weak affinity and poor therapeutic efficacy. No significant toxicities or adverse responses were observed. In conclusion, we demonstrated that rBC2LCN lectin binds CRC cells and that rBC2LCN‐PE38 significantly suppresses tumor growth in vivo. In addition, the efficacy of the drug conjugate correlated with its binding affinity for each CRC cell line. These results suggest that lectin drug conjugate therapy has potential as a novel targeted therapy for CRC cell surface glycans.

Repurposing Potential of Diminazene Aceturate as an Inhibitor of the E. coli DNA Gyrase B

Drug-resistant Escherichia coli (E. coli) has overburdened the healthcare facilities in recent years and is getting hard to combat, mandating search for novel therapeutics with a broad antibacterial spectrum and high chemotherapeutic index. The 24 kDa domain of DNA gyrase B that is involved in the ATPase activity has been reported to be a promising target for inhibitors. A PDB structure (1KZN) of the 24kD domain of gyrase B with the co-crystallized ligand clorobiocin was used for the docking studies to explore a library of 2924 FDA approved drugs from www.zinc.docking.org. FlexX docking module from Biosolve IT was used for receptor preparation and in silico docking experiments. Docking studies on the pocket created around the reference ligand clorobiocin revealed the best score with diminazene aceturate and it also demonstrated interactions with the crucial amino acids present within the pocket. Diminazene aceturate has been conventionally been used as an antiparasitic molecule in animals and it has also been demonstrated to exhibit repurposing potential in the treatment of disorders triggered due to overproduction of inflammatory cytokines, pulmonary hypertension, ischemia-induced cardiac pathophysiology, etc. among others. Findings from this study indicate the possibility of repurposing the age-old molecule diminazene aceturate into a DNA gyrase B antagonist to combat not just the drug-resistant E. coli but also other gram-negative ESKAPE pathogens. It may also aid in alleviating the inflammatory response induced in the body of the patients suffering from septicemia caused by a variety of Gram-negative bacterial pathogens.

De novo transcriptome analysis of Rhizophora mucronata Lam. furnishes evidence for the existence of glyoxalase system correlated to glutathione metabolic enzymes and glutathione regulated transporter in salt tolerant mangroves

The accumulation of a metabolic by product – methylglyoxal above a minimal range can be highly toxic in all organisms. Stress induced elevation in methylglyoxal inactivates proteins and nucleic acids. Glutathione dependent glyoxalase enzymes like glyoxalase I and glyoxalase II together with glutathione independent glyoxalase III play inevitable role in methylglyoxal detoxification. Glyoxalase genes are generally conserved but with obvious exceptions. Mangroves being potent harsh land inhabitants, their internal organelles are constantly been exposed to elevated levels of methylglyoxal. First and foremost it is important to detect the presence of glyoxalases in mangroves. De novo transcriptome analysis of mangrove species Rhizophora mucronata Lam., identified eleven putative glyoxalase proteins (RmGLYI-1 to 5, RmGLYII-1 to 5 and RmGLYIII). Molecular characterization proposed PLN02300 or PLN02367 as the key domains of RmGLYI proteins. They possess molecular weight ranging from 26.45 to 32.53 kDa and may localize in cytosol or chloroplast. RmGLYII proteins of molecular weight 28.64–36 kDa, carrying PLN02398 or PLN02469 domains are expected to be localized in diverse cellular compartments. Cytosolic RmGLYIII with DJ-1/PfpI domain carries a molecular weight 26.4 kDa. Detailed structural analysis revealed monomeric nature of RmGLYI-1 and RmGLYII-1 whereas RmGLYIII is found to be homodimer. Molecular phylogenetic analysis and multiple sequence alignment specified conserved metal ion/substrate binding residues of RmGLY proteins. Estimation of relative expression of glyoxalases under salt stress indicated the prominence of RmGLYI and RmGLYII over RmGLYIII. The aforementioned prominence is supported by salt induced expression difference of glutathione metabolic enzymes and glutathione regulated transporter protein.

Synthesis, molecular docking studies, and in vitro evaluation of 1,3,5-triazine derivatives as promising antimicrobial agents

The six-membered ring heterocycle 1,3,5-triazine and its derivatives have attracted considerable attention as they have proven to be excellent bioactive herbicides, cancer agents, etc. A series of 1,3,5-triazine derivatives (3a-o) were synthesized by a single step reaction and characterized by 1H NMR, 13C NMR, and mass spectrometry analysis. Antimicrobial screening of title compounds (3a-o) was examined against five bacterial and two fungal strains. In vitro study revealed that the freshly synthesized 6-(thiazol-4-yl)-1,3,5-triazine-2,4-diamine (3o) showed good antibacterial growth inhibition against E. coli, K. pneumoniae, and A. baumannii bacterial strains, and even the fungi C. neoformans. Molecular docking studies were performed on the X-ray crystal structure of E. coli 24 kDa domain in complex with clorobiocin (PDB code: 1KZN; resolution 2.30 Å) using Surflex-Dock program of Sybyl-X software. The results obtained are very encouraging.

Modeling of a 14 kDa RUVBL2-Binding Domain with Medium Resolution Cryo-EM Density

The number of high-resolution structures of protein complexes obtained using cryo-electron microscopy (cryo-EM) is increasing rapidly. Cryo-EM maps of large macromolecular complexes frequently contain regions resolved at different resolution levels, and modeling atomic structures de novo can be difficult for domains determined at worse than 5 Å in the absence of atomic information from other structures. Here we describe the details and step-by-step decisions in the strategy we followed to model the RUVBL2-binding domain (RBD), a 14 kDa domain at the C-terminus of RNA Polymerase II associated protein 3 (RPAP3) for which atomic information was not available. Modeling was performed on a cryo-EM map at 4.0-5.5 Å resolution, integrating information from secondary structure predictions, homology modeling, restraints from cross-linked mass spectrometry, and molecular dynamics (MD) in AMBER. Here, we compare our model with the structure of RBD determined by NMR to evaluate our strategy. We also perform new MD simulations to describe important residues mediating the interaction of RBD with RUVBL2 and analyze their conservation in RBD homologous domains. Our approach and its evaluation can serve as an example to address the analysis of medium resolution regions in cryo-EM maps.

Fluorescent Study on Tetanus Neurotoxin

Bacteria produce and secrete various toxins under specific circumstances. One class of these bacterial toxins, including the clostridial neurotoxins, are a special class of proteins which begin in a globular, soluble form and convert to an integral membrane protein. The clostridial neurotoxins have a three globular domain structure with each portion responsible for one aspect of its function. During intoxication, it inserts into the endosomal membrane and concurrently translocates a portion of the protein into the mammalian cytosol, where it has toxic activities. The middle 50kDa domain is the translocation domain. This domain is responsible for the switch from globular soluble to integral membrane and translocating a second domain across the endosomal membrane, in response to the acidic low pH of the endosomal lumen. However, the exact residues which enter the membrane are not known. By putting an environmentally sensitive fluorescent probe at various positions and controlling the environment through the addition of membranes and then low pH, the local environment of each position can be monitored. From our studies, we have identified residues which are membrane inserted and further differentiated their depth into the membrane bilayer.

Bacterial sugar-binding protein as a one-step affinity purification tag on dextran-containing resins

Marinobacter hydrocarbonoclasticus is an oil-eating bacterium that possesses a large adhesion protein (MhLap) with the potential to bind extracellular ligands. One of these ligand-binding modules is the ~20-kDa PA14 domain (MhPA14) that has affinity for glucose-based carbohydrates. Previous studies showed this sugar-binding domain is retained on dextran-based size-exclusion resins during chromatography, requiring the introduction of glucose or EDTA to remove the protein from the column. Given the ready availability of such size-exclusion resins in biochemistry laboratories, this study explores the use of MhPA14 as an affinity tag for recombinant protein purification. Two different fusion proteins were tested: 1) Green fluorescent protein (GFP) linked to the N-terminus of the MhPA14 tag; and 2) the ice-binding domain from the Marinomonas primoryensis ice-binding protein (MpIBD) linked to the MhPA14 C-terminus by a TEV cut site. The GFP_MhPA14 fusion visibly bound to Superdex, Sephadex, and Sephacryl resins, but did not bind to Sepharose. Using Superdex resin, dextran-affinity purification proved to be an effective one-step purification strategy for both proteins, superior to even nickel-affinity chromatography. Dextran-affinity chromatography was also the most effective method of separating the MhPA14 tag from MpIBD following TEV proteolysis, as compared to both nickel-affinity and ice-affinity methods. These results indicate that MhPA14 has potential for widespread use in recombinant protein purification.

Synthesis, characterization and pharmacological evaluation of 2-aminothiazole incorporated azo dyes

Azo molecules (A1-A3) were prepared by reacting 2-aminothiazole with three different pyridone derivatives as coupling components, viz., 6-hydroxy-4-methyl-2-oxo-1,2-dihydropyridine-3-carbonitrile, 6-hydroxy-1,4-dimethyl-2-oxo-1,2-dihydropyridine-3-nitrile and 1-ethyl-6-hydroxy-4-methyl-2-oxo-1,2-dihydropyridine-3-carbonitrile via diazo-coupling reactions, under appropriate experimental conditions. The structural characteristics of the synthesized compounds were checked by different spectral techniques. The newly synthesized molecules were tested for their biological activities and the results of biological activity studies of the compounds prepared exhibited potential antimicrobial activity against the respective microorganisms. The synthesized azo dyes also demonstrated promising antimycobacterial activity screened against M. tuberculosis. All the compounds showed considerable cleavage efficiency against supercoiled pBR322 DNA. Further, the in silico molecular docking study was performed against E. coli 24 kDa domain and Cyp51 (cytochrome P450 14α-sterol demethylase) as target receptors. The binding energy of each studied compound was evaluated, and the data revealed that the compound A1 displayed higher binding affinity value and forms hydrogen bonds with the active amino acids.

Structure and functional analysis of a bacterial adhesin sugar-binding domain.

Bacterial adhesins attach their hosts to surfaces through one or more ligand-binding domains. In RTX adhesins, which are localized to the outer membrane of many Gram-negative bacteria via the type I secretion system, we see several examples of a putative sugar-binding domain. Here we have recombinantly expressed one such ~20-kDa domain from the ~340-kDa adhesin found in Marinobacter hydrocarbonoclasticus, an oil-degrading bacterium. The sugar-binding domain was purified from E. coli with a yield of 100 mg/L of culture. Circular dichroism analysis showed that the protein was rich in beta-structure, was moderately heat resistant, and required Ca2+ for proper folding. A crystal structure was obtained in Ca2+ at 1.2-Å resolution, which showed the presence of three Ca2+ ions, two of which were needed for structural integrity and one for binding sugars. Glucose was soaked into the crystal, where it bound to the sugar’s two vicinal hydroxyl groups attached to the first and second (C1 and C2) carbons in the pyranose ring. This attraction to glucose caused the protein to bind certain polysaccharide-based column matrices and was used in a simple competitive binding assay to assess the relative affinity of sugars for the protein’s ligand-binding site. Fucose, glucose and N-acetylglucosamine bound most tightly, and N-acetylgalactosamine hardly bound at all. Isothermal titration calorimetry was used to determine specific binding affinities, which lie in the 100-μM range. Glycan arrays were tested to expand the range of ligand sugars assayed, and showed that MhPA14 bound preferentially to branched polymers containing terminal sugars highlighted as strong binders in the competitive binding assay. Some of these binders have vicinal hydroxyl groups attached to the C3 and C4 carbons that are sterically equivalent to those presented by the C1 and C2 carbons of glucose.