Recombinant Photoproteins in the Design of Binding Assays

AbstractLuminescent CdSe-ZnS core-shell quantum dot (QD) bioconjugates were used as energy donors in fluorescent resonance energy transfer (FRET) binding assays. The QDs were coated with saturating amounts of genetically engineered maltose binding protein (MBP) using a noncovalent immobilization process, and Cy3 organic dyes covalently attached at a specific sequence to MBP were used as energy acceptor molecules. Energy transfer efficiency was measured as a function of the MBP-Cy3/QD molar ratio for two different donor fluorescence emissions (different QD core sizes). Apparent donor-acceptor distances were determined from these FRET studies, and the measured distances are consistent with QD-protein conjugate dimensions previously determined from structural studies.

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Single Amino Acid Based Self-Assemblies of Cysteine and Methionine

10.26434/chemrxiv.5972173.v1 ◽

2018 ◽

Author(s):

Nidhi Gour ◽

Bharti Koshti ◽

Chandra Kanth P. ◽

Dhruvi Shah ◽

Vivek Shinh Kshatriya ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Self Assembly ◽

Aromatic Amino Acids ◽

Neutral Condition ◽

Single Amino Acid ◽

Binding Assays ◽

Human Neuroblastoma ◽

Cytotoxicity Assays ◽

First Time

We report for the very first time self-assembly of Cysteine and Methionine to discrenible strucutres under neutral condition. To get insights into the structure formation, thioflavin T and Congo red binding assays were done which revealed that aggregates may not have amyloid like characteristics. The nature of interactions which lead to such self-assemblies was purported by coincubating assemblies in urea and mercaptoethanol. Further interaction of aggregates with short amyloidogenic dipeptide diphenylalanine (FF) was assessed. While cysteine aggregates completely disrupted FF fibres, methionine albeit triggered fibrillation. The cytotoxicity assays of cysteine and methionine structures were performed on Human Neuroblastoma IMR-32 cells which suggested that aggregates are not cytotoxic in nature and thus, may not have amyloid like etiology. The results presented in the manuscript are striking, since to the best of our knowledge,this is the first report which demonstrates that even non-aromatic amino acids (cysteine and methionine) can undergo spontaneous self-assembly to form ordered aggregates.

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Faculty Opinions recommendation of Tuning the pK(a) of fluorescein to optimize binding assays.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1090152.543368 ◽

2007 ◽

Author(s):

Nicola Pohl

Keyword(s):

Binding Assays

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Identification of a Coronavirus Hemagglutinin-Esterase with a Substrate Specificity Different from Those of Influenza C Virus and Bovine Coronavirus

Journal of Virology ◽

10.1128/jvi.73.5.3737-3743.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 3737-3743 ◽

Cited By ~ 46

Author(s):

Alfred Klausegger ◽

Birgit Strobl ◽

Gerhard Regl ◽

Alexandra Kaser ◽

Willem Luytjes ◽

...

Keyword(s):

Substrate Specificity ◽

Solid Phase ◽

Hepatitis Virus ◽

Bovine Brain ◽

Brain Extract ◽

Binding Assays ◽

Bovine Coronavirus ◽

Close Relationship ◽

Influenza C Virus ◽

Cloned Gene

ABSTRACT We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substratesp-nitrophenyl acetate, α-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse α1macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.

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Novel Transcriptional Regulons for Autotrophic Cycle Genes in Crenarchaeota

Journal of Bacteriology ◽

10.1128/jb.00249-15 ◽

2015 ◽

Vol 197 (14) ◽

pp. 2383-2391 ◽

Cited By ~ 7

Author(s):

Semen A. Leyn ◽

Irina A. Rodionova ◽

Xiaoqing Li ◽

Dmitry A. Rodionov

Keyword(s):

Carbon Dioxide ◽

Transcriptional Regulation ◽

Comparative Genomics ◽

Dna Binding ◽

Transcriptional Control ◽

Binding Assays ◽

Promoter Regions ◽

Content Type ◽

Genome Context

ABSTRACTAutotrophic microorganisms are able to utilize carbon dioxide as their only carbon source, or, alternatively, many of them can grow heterotrophically on organics. Different variants of autotrophic pathways have been identified in various lineages of the phylumCrenarchaeota. Aerobic members of the orderSulfolobalesutilize the hydroxypropionate-hydroxybutyrate cycle (HHC) to fix inorganic carbon, whereas anaerobicThermoprotealesuse the dicarboxylate-hydroxybutyrate cycle (DHC). Knowledge of transcriptional regulation of autotrophic pathways inArchaeais limited. We applied a comparative genomics approach to predict novel autotrophic regulons in theCrenarchaeota. We report identification of two novel DNA motifs associated with the autotrophic pathway genes in theSulfolobales(HHC box) andThermoproteales(DHC box). Based on genome context evidence, the HHC box regulon was attributed to a novel transcription factor from the TrmB family named HhcR. Orthologs of HhcR are present in allSulfolobalesgenomes but were not found in other lineages. A predicted HHC box regulatory motif was confirmed byin vitrobinding assays with the recombinant HhcR protein fromMetallosphaera yellowstonensis. For the DHC box regulon, we assigned a different potential regulator, named DhcR, which is restricted to the orderThermoproteales. DhcR inThermoproteus neutrophilus(Tneu_0751) was previously identified as a DNA-binding protein with high affinity for the promoter regions of two autotrophic operons. The global HhcR and DhcR regulons reconstructed by comparative genomics were reconciled with available omics data inMetallosphaeraandThermoproteusspp. The identified regulons constitute two novel mechanisms for transcriptional control of autotrophic pathways in theCrenarchaeota.IMPORTANCELittle is known about transcriptional regulation of carbon dioxide fixation pathways inArchaea. We previously applied the comparative genomics approach for reconstruction of DtxR family regulons in diverse lineages ofArchaea. Here, we utilize similar computational approaches to identify novel regulatory motifs for genes that are autotrophically induced in microorganisms from two lineages ofCrenarchaeotaand to reconstruct the respective regulons. The predicted novel regulons in archaeal genomes control the majority of autotrophic pathway genes and also other carbon and energy metabolism genes. The HhcR regulon was experimentally validated by DNA-binding assays inMetallosphaeraspp. Novel regulons described for the first time in this work provide a basis for understanding the mechanisms of transcriptional regulation of autotrophic pathways inArchaea.

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Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

Biochemical Journal ◽

10.1042/bj20080234 ◽

2008 ◽

Vol 415 (1) ◽

pp. 27-33 ◽

Cited By ~ 45

Author(s):

Meghna Thakur ◽

Pradip K. Chakraborti

Keyword(s):

Protein Kinases ◽

Solid Phase ◽

Morphological Changes ◽

Binding Assays ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Peptidoglycan Biosynthesis ◽

Vitro Binding ◽

Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

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An epilepsy-causing mutation leads to co-translational misfolding of the Kv7.2 channel

BMC Biology ◽

10.1186/s12915-021-01040-1 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Janire Urrutia ◽

Alejandra Aguado ◽

Carolina Gomis-Perez ◽

Arantza Muguruza-Montero ◽

Oscar R. Ballesteros ◽

...

Keyword(s):

Structural Instability ◽

Binding Assays ◽

Iq Motif ◽

Channel Function ◽

Human Epilepsy ◽

Pathogenic Variants ◽

In Silico Studies ◽

Nascent Chain

Abstract Background The amino acid sequence of proteins generally carries all the necessary information for acquisition of native conformations, but the vectorial nature of translation can additionally determine the folding outcome. Such consideration is particularly relevant in human diseases associated to inherited mutations leading to structural instability, aggregation, and degradation. Mutations in the KCNQ2 gene associated with human epilepsy have been suggested to cause misfolding of the encoded Kv7.2 channel. Although the effect on folding of mutations in some domains has been studied, little is known of the way pathogenic variants located in the calcium responsive domain (CRD) affect folding. Here, we explore how a Kv7.2 mutation (W344R) located in helix A of the CRD and associated with hereditary epilepsy interferes with channel function. Results We report that the epilepsy W344R mutation within the IQ motif of CRD decreases channel function, but contrary to other mutations at this site, it does not impair the interaction with Calmodulin (CaM) in vitro, as monitored by multiple in vitro binding assays. We find negligible impact of the mutation on the structure of the complex by molecular dynamic computations. In silico studies revealed two orientations of the side chain, which are differentially populated by WT and W344R variants. Binding to CaM is impaired when the mutated protein is produced in cellulo but not in vitro, suggesting that this mutation impedes proper folding during translation within the cell by forcing the nascent chain to follow a folding route that leads to a non-native configuration, and thereby generating non-functional ion channels that fail to traffic to proper neuronal compartments. Conclusions Our data suggest that the key pathogenic mechanism of Kv7.2 W344R mutation involves the failure to adopt a configuration that can be recognized by CaM in vivo but not in vitro.

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A Reversible Shift of Driver Dependence from EGFR to Notch1 in Non-Small Cell Lung Cancer as a Cause of Resistance to Tyrosine Kinase Inhibitors

Cancers ◽

10.3390/cancers13092022 ◽

2021 ◽

Vol 13 (9) ◽

pp. 2022

Author(s):

Francesca Iommelli ◽

Viviana De Rosa ◽

Cristina Terlizzi ◽

Rosa Fonti ◽

Rosa Camerlingo ◽

...

Keyword(s):

Kinase Inhibitors ◽

Epithelial Mesenchymal Transition ◽

Parental Cell ◽

Egfr Inhibitors ◽

Simultaneous Increase ◽

Adherent Cells ◽

Binding Assays ◽

Mesenchymal Transition ◽

Dose Dependent Decrease ◽

Dose Dependent

Notch1 plays a key role in epithelial-mesenchymal transition (EMT) and in the maintenance of cancer stem cells. In the present study we tested whether high levels of activated Notch1 in oncogene-driven NSCLC can induce a reversible shift of driver dependence from EGFR to Notch1, and thus causing resistance to EGFR inhibitors. Adherent cells (parental) and tumor spheres (TS) from NSCLC H1975 cells and patient-derived CD133-positive cells were tested for EGFR and Notch1 signaling cascade. The Notch1-dependent modulation of EGFR, NCID, Hes1, p53, and Sp1 were then analyzed in parental cells by binding assays with a Notch1 agonist, DLL4. TS were more resistant than parental cells to EGFR inhibitors. A strong upregulation of Notch1 and a concomitant downregulation of EGFR were observed in TS compared to parental cells. Parental cell exposure to DLL4 showed a dose-dependent decrease of EGFR and a simultaneous increase of NCID, Hes1, p53, and Sp1, along with the dislocation of Sp1 from the EGFR promoter. Furthermore, an enhanced interaction between p53 and Sp1 was observed in TS. In NSCLC cells, high levels of active Notch1 can promote a reversible shift of driver dependence from EGFR to Notch1, leading to resistance to EGFR inhibitors.

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The structure of SeviL, a GM1b/asialo-GM1 binding R-type lectin from the mussel Mytilisepta virgata

Scientific Reports ◽

10.1038/s41598-020-78926-7 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Kenichi Kamata ◽

Kenji Mizutani ◽

Katsuya Takahashi ◽

Roberta Marchetti ◽

Alba Silipo ◽

...

Keyword(s):

Immune System ◽

Sialic Acid ◽

Ligand Binding ◽

Steric Hindrance ◽

Sequence Similarity ◽

Binding Assays ◽

Subunit Interface ◽

Nmr Techniques ◽

And Control ◽

Asialo Gm1

AbstractSeviL is a recently isolated lectin found to bind to the linear saccharides of the ganglioside GM1b (Neu5Ac$$\alpha$$ α (2-3)Gal$$\beta$$ β (1-3)GalNAc$$\beta$$ β (1-4)Gal$$\beta$$ β (1-4)Glc) and its precursor, asialo-GM1 (Gal$$\beta$$ β (1-3)GalNAc$$\beta$$ β (1-4)Gal$$\beta$$ β (1-4)Glc). The crystal structures of recombinant SeviL have been determined in the presence and absence of ligand. The protein belongs to the $$\beta$$ β -trefoil family, but shows only weak sequence similarity to known structures. SeviL forms a dimer in solution, with one binding site per subunit, close to the subunit interface. Molecular details of glycan recognition by SeviL in solution were analysed by ligand- and protein-based NMR techniques as well as ligand binding assays. SeviL shows no interaction with GM1 due to steric hindrance with the sialic acid branch that is absent from GM1b. This unusual specificity makes SeviL of great interest for the detection and control of certain cancer cells, and cells of the immune system, that display asialo-GM1.

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Use of Half-Generation PAMAM Dendrimers (G0.5–G3.5) with Carboxylate End-Groups to Improve the DACHPtCl2 and 5-FU Efficacy as Anticancer Drugs

Molecules ◽

10.3390/molecules26102924 ◽

2021 ◽

Vol 26 (10) ◽

pp. 2924

Author(s):

Cláudia Camacho ◽

Helena Tomás ◽

João Rodrigues

Keyword(s):

Cancer Cells ◽

Cell Lines ◽

Cancer Cell ◽

Anticancer Drug ◽

Water Solubility ◽

Cancer Cell Lines ◽

Pamam Dendrimers ◽

Binding Assays ◽

End Groups ◽

Ic50 Values

The DACHPtCl2 compound (trans-(R,R)-1,2-diaminocyclohexanedichloroplatinum(II)) is a potent anticancer drug with a broad spectrum of activity and is less toxic than oxaliplatin (trans-l-diaminocyclohexane oxalate platinum II), with which it shares the active metal fragment DACHPt. Nevertheless, due to poor water solubility, its use as a chemotherapeutic drug is limited. Here, DACHPtCl2 was conjugated, in a bidentate form, with half-generation PAMAM dendrimers (G0.5–G3.5) with carboxylate end-groups, and the resulting conjugates were evaluated against various types of cancer cell lines. In this way, we aimed at increasing the solubility and availability at the target site of DACHPt while potentially reducing the adverse side effects. DNA binding assays showed a hyperchromic effect compatible with DNA helix’s disruption upon the interaction of the metallodendrimers and/or the released active metallic fragments with DNA. Furthermore, the prepared DACHPt metallodendrimers presented cytotoxicity in a wide set of cancer cell lines used (the relative potency regarding oxaliplatin was in general high) and were not hemotoxic. Importantly, their selectivity for A2780 and CACO-2 cancer cells with respect to non-cancer cells was particularly high. Subsequently, the anticancer drug 5-FU was loaded in a selected metallodendrimer (the G2.5COO(DACHPt)16) to investigate a possible synergistic effect between the two drugs carried by the same dendrimer scaffold and tested for cytotoxicity in A2780cisR and CACO-2 cancer cell lines. This combination resulted in IC50 values much lower than the IC50 for 5-FU but higher than those found for the metallodendrimers without 5-FU. It seems, thus, that the metallic fragment-induced cytotoxicity dominates over the cytotoxicity of 5-FU in the set of considered cell lines.

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