scholarly journals Single-molecule kinetic locking allows fluorescence-free quantification of protein/nucleic-acid binding

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Martin Rieu ◽  
Jessica Valle-Orero ◽  
Bertrand Ducos ◽  
Jean-François Allemand ◽  
Vincent Croquette
Keyword(s):  
Single Molecule ◽  
Functional Characterization ◽  
Nucleic Acid Binding ◽  
Single Molecule Force Spectroscopy ◽  
E Coli ◽  
Natural Function ◽  
Protein Nucleic Acid ◽  
Free Quantification

AbstractFluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5, 6, 7 bases) and use it to measure the dynamical interactions of Escherichia coli/E. coli RecQ helicase with its DNA substrate.

scholarly journals Fluorescence-free quantification of protein/nucleic-acid binding through single-molecule kinetic locking

2020 ◽  
Author(s):  
Martin Rieu ◽  
Jessica Valle-Orero ◽  
Bertrand Ducos ◽  
Jean-François Allemand ◽  
Vincent Croquette
Keyword(s):  
Single Molecule ◽  
Functional Characterization ◽  
Nucleic Acid Binding ◽  
Single Molecule Force Spectroscopy ◽  
E Coli ◽  
Natural Function ◽  
Protein Nucleic Acid ◽  
Free Quantification

ABSTRACTFluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a new method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5,6,7 bases) and use it to measure the dynamical interactions of E. coli RecQ helicase with its DNA substrate.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Ferric Enterochelin Transport in Yersinia enterocolitica: Molecular and Evolutionary Aspects

1999 ◽  
Vol 181 (20) ◽  
pp. 6387-6395 ◽  
Author(s):  
S. Schubert ◽  
D. Fischer ◽  
J. Heesemann
Keyword(s):  
Gene Cluster ◽  
Yersinia Enterocolitica ◽  
Insertional Mutagenesis ◽  
Yersinia Pseudotuberculosis ◽  
Functional Characterization ◽  
In Vitro Transcription ◽  
E Coli ◽  
The Family

ABSTRACT Yersinia enterocolitica is well equipped for siderophore piracy, encompassing the utilization of siderophores such as ferrioxamine, ferrichrome, and ferrienterochelin. In this study, we report on the molecular and functional characterization of theYersinia fep-fes gene cluster orthologous to theEscherichia coli ferrienterochelin transport genes (fepA, fepDGC, and fepB) and the esterase gene fes. In vitro transcription-translation analysis identified polypeptides of 30 and 35 kDa encoded byfepC and fes, respectively. A frameshift mutation within the fepA gene led to expression of a truncated polypeptide of 40 kDa. The fepD,fepG, and fes genes of Y. enterocolitica were shown to complement corresponding E. coli mutants. Insertional mutagenesis of fepD orfes genes abrogates enterochelin-supported growth ofY. enterocolitica on iron-chelated media. In contrast toE. coli, the fep-fes gene cluster inY. enterocolitica consists solely of genes required for uptake and utilization of enterochelin (fep) and not of enterochelin synthesis genes such as entF. By Southern hybridization, fepDGC and fes sequences could be detected in Y. enterocolitica biotypes IB, IA, and II but not in biotype IV strains, Yersinia pestis, andYersinia pseudotuberculosis strains. According to sequence alignment data and the coherent structure of the Yersinia fep-fes gene cluster, we suggest early genetic divergence of ferrienterochelin uptake determinants among species of the familyEnterobacteriaceae.

scholarly journals cfrB, cfrC, and a potential new cfr-like gene in Clostridium difficile strains recovered across Latin America

2019 ◽  
Author(s):  
Vanja Stojković ◽  
María Fernanda Ulate ◽  
Fanny Hidalgo-Villeda ◽  
Emmanuel Aguilar ◽  
Camilo Monge-Cascante ◽  
...  
Keyword(s):  
Latin America ◽  
Ribosomal Proteins ◽  
Functional Characterization ◽  
Ribosomal Subunit ◽  
23S Rrna ◽  
Cross Resistance ◽  
Protection Factor ◽  
E Coli

ABSTRACTCfr is a radical S-adenosyl-L-methionine (SAM) enzyme that confers cross-resistance to all antibiotics targeting the large ribosomal subunit through hypermethylation of nucleotide A2503 of 23S rRNA. Of the four known cfr genes known to date, cfr(B) and cfr(C) have been sporadically found in C. difficile, yet functional characterization of cfr(C) is still lacking. We identified genes for putative Cfr-like enzymes among clinical C. difficile strains from Mexico, Honduras, Costa Rica, and Chile. To confirm their identity and activity, we obtained minimum inhibitory concentrations for ribosome-targeting antibiotics, annotated whole genome sequences, and performed a functional characterization of Cfr(C). The seven representative isolates analyzed displayed different levels of resistance to PhLOPSA antibiotics in the absence of the ribosome protection factor OptrA, and mutations in genes for 23S rRNAs or the ribosomal proteins L3 and L4. cfr(B) was detected in four isolates as part of a Tn6218-like transposon or an un-described mobile genetic element. In turn, cfr(C) was found integrated into an ICE-element. One isolate harbored a putative cfr-like gene that shows only 51-58% of sequence identity to Cfr and known Cfr-like enzymes. Moreover, our in vitro assays confirmed that Cfr(C) methylates E. coli and C. difficile 23S rRNA fragments. These results indicate selection of cfr-like genes in C. difficile from Latin America, suggest that the diversity of cfr-like resistance genes is larger than anticipated, and provide the first assessment of the methylation activity of Cfr(C).

scholarly journals Functional Characterization of POFUT1 Variants Associated with Colorectal Cancer

Cancers ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1430 ◽  
Author(s):  
Marlène Deschuyter ◽  
Florian Pennarubia ◽  
Emilie Pinault ◽  
Sébastien Legardinier ◽  
Abderrahman Maftah
Keyword(s):  
Colorectal Cancer ◽  
Notch Signaling ◽  
Functional Characterization ◽  
Missense Mutations ◽  
Colorectal Tumors ◽  
Protein Targets ◽  
E Coli ◽  
Notch Receptors

Background: Protein O-fucosyltransferase 1 (POFUT1) overexpression, which is observed in many cancers such as colorectal cancer (CRC), leads to a NOTCH signaling dysregulation associated with the tumoral process. In rare CRC cases, with no POFUT1 overexpression, seven missense mutations were found in human POFUT1. Methods: Recombinant secreted forms of human WT POFUT1 and its seven mutated counterparts were produced and purified. Their O-fucosyltransferase activities were assayed in vitro using a chemo-enzymatic approach with azido-labeled GDP-fucose as a donor substrate and NOTCH1 EGF-LD26, produced in E. coli periplasm, as a relevant acceptor substrate. Targeted mass spectrometry (MS) was carried out to quantify the O-fucosyltransferase ability of all POFUT1 proteins. Findings: MS analyses showed a significantly higher O-fucosyltransferase activity of six POFUT1 variants (R43H, Y73C, T115A, I343V, D348N, and R364W) compared to WT POFUT1. Interpretation: This study provides insights on the possible involvement of these seven missense mutations in colorectal tumors. The hyperactive forms could lead to an increased O-fucosylation of POFUT1 protein targets such as NOTCH receptors in CRC patients, thereby leading to a NOTCH signaling dysregulation. It is the first demonstration of gain-of-function mutations for this crucial glycosyltransferase, modulating NOTCH activity, as well as that of other potential glycoproteins.

scholarly journals Sweet Basil Has Distinct Synthases for Eugenol Biosynthesis in Glandular Trichomes and Roots with Different Regulatory Mechanisms

2021 ◽  
Vol 22 (2) ◽  
pp. 681
Author(s):  
Vaishnavi Amarr Reddy ◽  
Chunhong Li ◽  
Kumar Nadimuthu ◽  
Jessica Gambino Tjhang ◽  
In-Cheol Jang ◽  
...  
Keyword(s):  
Glandular Trichomes ◽  
Functional Characterization ◽  
Rna Seq ◽  
Post Translational Modifications ◽  
E Coli ◽  
Sweet Basil ◽  
Specific Regulation

Production of a volatile phenylpropene; eugenol in sweet basil is mostly associated with peltate glandular trichomes (PGTs) found aerially. Currently only one eugenol synthase (EGS), ObEGS1 which belongs to PIP family is identified from sweet basil PGTs. Reports of the presence of eugenol in roots led us to analyse other EGSs in roots. We screened for all the PIP family reductase transcripts from the RNA-Seq data. In vivo functional characterization of all the genes in E. coli showed their ability to produce eugenol and were termed as ObEGS2-8. Among all, ObEGS1 displayed highest expression in PGTs and ObEGS4 in roots. Further, eugenol was produced only in the roots of soil-grown plants, but not in roots of aseptically-grown plants. Interestingly, eugenol production could be induced in roots of aseptically-grown plants under elicitation suggesting that eugenol production might occur as a result of environmental cues in roots. The presence of ObEGS4 transcript and protein in aseptically-grown plants indicated towards post-translational modifications (PTMs) of ObEGS4. Bioinformatics analysis showed possibility of phosphorylation in ObEGS4 which was further confirmed by in vitro experiment. Our study reveals the presence of multiple eugenol synthases in sweet basil and provides new insights into their diversity and tissue specific regulation.

scholarly journals Efficient in vitro refolding and functional characterization of recombinant human liver carboxylesterase (CES1) expressed in E. coli

2015 ◽  
Vol 107 ◽  
pp. 68-75 ◽  
Author(s):  
Usa Boonyuen ◽  
Kamoltip Promnares ◽  
Suwapat Junkree ◽  
Nichloas P.J. Day ◽  
Mallika Imwong
Keyword(s):  
Human Liver ◽  
Functional Characterization ◽  
E Coli ◽  
In Vitro Refolding

Prokaryotic functional expression and activity comparison of three CYP9A genes from the polyphagous pestHelicoverpa armigera

2017 ◽  
Vol 108 (1) ◽  
pp. 77-83 ◽  
Author(s):  
D. Liu ◽  
K. Tian ◽  
Y. Yuan ◽  
M. Li ◽  
M. Zheng ◽  
...  
Keyword(s):  
Pyrethroid Resistance ◽  
Functional Characterization ◽  
Kinetic Studies ◽  
Functional Expression ◽  
Cytochrome P450 Reductase ◽  
E Coli ◽  
Important Pest ◽  
Activity Comparison

AbstractCytochrome P450s (CYPs or P450s) have been long recognized as very important enzymes in the metabolism of xenobiotic and endogenous compounds, but only a few CYPs have been functionally characterized in insects. The effort in functional characterization of insect P450s is heavily hindered by technical difficulties in preparing active, individual P450 enzymes directly from the target insect. In this paper, we describe the functional expression of two additional pyrethroid resistance-associated CYP9A genes (CYP9A12andCYP9A17) from the polyphagous pestHelicoverpa armigerain the facileEscherichia coli. The functionality ofE. coliproduced CYP9A12, CYP9A14, and CYP9A17 was investigated and activities of these CYP9As were compared against three probe substrates after reconstitution with NADPH-dependent cytochrome P450 reductase. The results showed that active forms of CYP9A12 and CYP9A17 were expressed inE. coliwith a content of about 1.0–1.5 nmol mg−1protein in membrane preparations.In vitroassays showed that CYP9A14 was capable of catalyzing O-dealkylation of methoxyresorufin (MROD), ethoxyresorufin (EROD), and benzyloxyresorufin (BROD), while CYP9A12 and CYP9A17 exhibited only MROD and EROD activities. Kinetic studies demonstrated that CYP9A14 had the greatestkcat/Kmvalue for MROD, and CYP9A17 for EROD, while the lowestkcat/Kmvalues for both MROD and EROD were observed for CYP9A12. The distinct biochemical traits suggest that the three paralogous CYP9As may play different roles in xenobiotic metabolism in this important pest.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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