Biophysical Characterization of Interaction between E. coli Alanyl-tRNA Synethase with its Promoter DNA

2020 ◽  
Vol 27 (7) ◽  
pp. 635-648
Author(s):  
Baisakhi Banerjee ◽  
Sayak Ganguli ◽  
Rajat Banerjee
Keyword(s):  
Dna Binding ◽  
Analytical Ultracentrifugation ◽  
Isothermal Calorimetry ◽  
Trna Synthetase ◽  
Full Length ◽  
Sedimentation Equilibrium ◽  
Titration Calorimetry ◽  
E Coli ◽  
Promoter Dna

Background: Aminoacyl-tRNA Synthetases (aaRSs) are well known for their role in the translation process. Lately investigators have discovered that this family of enzymes are also capable of executing a broad repertoire of functions that not only impact protein synthesis, but extend to a number of other activities. Till date, transcriptional regulation has so far only been described in E. coli Alanyl-tRNA synthetase and it was demonstrated that alaRS binds specifically to the palindromic DNA sequence flanking the gene’s transcriptional start site and thereby regulating its own transcription. Objective: In the present study, we have characterized some of the features of the alaRS-DNA binding using various biophysical techniques. Methods: To understand the role of full length protein and oligomerization of alaRS in promoter DNA binding, two mutants were constructed, namely, N700 (a monomer, containing the N-terminal aminoacylation domain but without the C-terminal part) and G674D (previously demonstrated to form full-length monomer). Protein-DNA binding study using fluorescence spectroscopy, analytical ultracentrifugation, Isothermal Titration Calorimetry was conducted. Results: Sedimentation equilibrium studies clearly demonstrated that monomeric variants were unable to bind promoter DNA. Isothermal Calorimetry (ITC) experiment was employed for further characterization of wild type alaRS-DNA interaction. It was observed that full length E. coli Alanyl-tRNA synthetase binds specifically with its promoter DNA and forms a dimer of dimers. On the other hand the two mutant variants were unable to bind with the DNA. Conclusion: In this study it was concluded that full length E. coli Alanyl-tRNA synthetase undergoes a conformational change in presence of its promoter DNA leading to formation of higher order structures. However, the exact mechanism behind this binding is currently unknown and beyond the scope of this study.

scholarly journals Urea Unfolding Study of E. coli Alanyl-tRNA Synthetase and Its Monomeric Variants Proves the Role of C-Terminal Domain in Stability

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Baisakhi Banerjee ◽  
Rajat Banerjee
Keyword(s):  
Analytical Ultracentrifugation ◽  
Coiled Coil ◽  
Tryptophan Fluorescence ◽  
Trna Synthetase ◽  
Full Length ◽  
Spectroscopic Techniques ◽  
Wild Type ◽  
Native Form ◽  
E Coli ◽  
Terminal Domain

E. coli alanyl-tRNA exists as a dimer in its native form and the C-terminal coiled-coil part plays an important role in the dimerization process. The truncated N-terminal containing the first 700 amino acids (1–700) forms a monomeric variant possessing similar aminoacylation activity like wild type. A point mutation in the C-terminal domain (G674D) also produces a monomeric variant with a fivefold reduced aminoacylation activity compared to the wild type enzyme. Urea induced denaturation of these monomeric mutants along with another alaRS variant (N461 alaRS) was studied together with the full-length enzyme using various spectroscopic techniques such as intrinsic tryptophan fluorescence, 1-anilino-8-naphthalene-sulfonic acid binding, near- and far-UV circular dichroism, and analytical ultracentrifugation. Aminoacylation activity assay after refolding from denatured state revealed that the monomeric mutants studied here were unable to regain their activity, whereas the dimeric full-length alaRS gets back similar activity as the native enzyme. This study indicates that dimerization is one of the key regulatory factors that is important in the proper folding and stability of E. coli alaRS.

Purification and functional characterization of the full length recombinant Ebola virus VP35 protein expressed in E. coli

2009 ◽  
Vol 66 (1) ◽  
pp. 113-119 ◽  
Author(s):  
Luca Zinzula ◽  
Francesca Esposito ◽  
Elke Mühlberger ◽  
Martina Trunschke ◽  
Dominik Conrad ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Functional Characterization ◽  
Full Length ◽  
E Coli

scholarly journals Enhanced oligomerization of full-length RAGE by synergy of the interaction of its domains

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Alexander Moysa ◽  
Dietmar Hammerschmid ◽  
Roman H. Szczepanowski ◽  
Frank Sobott ◽  
Michal Dadlez
Keyword(s):  
Analytical Ultracentrifugation ◽  
Vascular Diseases ◽  
Native Mass Spectrometry ◽  
Full Length ◽  
Size Exclusion ◽  
Glycation End Products ◽  
Exclusion Chromatography ◽  
Hydrogen Deuterium

AbstractThe pattern recognition receptor RAGE (receptor for advanced glycation end-products) transmits proinflammatory signals in several inflammation-related pathological states, including vascular diseases, cancer, neurodegeneration and diabetes. Its oligomerization is believed to be important in signal transduction, but RAGE oligomeric structures and stoichiometries remain unclear. Different oligomerization modes have been proposed in studies involving different truncated versions of the extracellular parts of RAGE. Here, we provide basic characterization of the oligomerization patterns of full-length RAGE (including the transmembrane (TM) and cytosolic regions) and compare the results with oligomerization modes of its four truncated fragments. For this purpose, we used native mass spectrometry, analytical ultracentrifugation, and size-exclusion chromatography coupled with multi-angle light scattering. Our results confirm known oligomerization tendencies of separate domains and highlight the enhanced oligomerization properties of full-length RAGE. Mutational analyses within the GxxxG motif of the TM region show sensitivity of oligomeric distributions to the TM sequence. Using hydrogen–deuterium exchange, we mapped regions involved in TM-dependent RAGE oligomerization. Our data provide experimental evidence for the major role of the C2 and TM domains in oligomerization, underscoring synergy among different oligomerization contact regions along the RAGE sequence. These results also explain the variability of obtained oligomerization modes in RAGE fragments.

scholarly journals Structural and Biophysical Characterization of the Proteins Interacting with the Herpes Simplex Virus 1 Origin of Replication

2009 ◽  
Vol 284 (24) ◽  
pp. 16343-16353 ◽  
Author(s):  
Ioannis Manolaridis ◽  
Eleni Mumtsidu ◽  
Peter Konarev ◽  
Alexander M. Makhov ◽  
Stephen W. Fullerton ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dna Binding ◽  
Analytical Ultracentrifugation ◽  
Binding Protein ◽  
Size Exclusion ◽  
Titration Calorimetry ◽  
Origin Of Replication ◽  
Mobility Shift ◽  
Simplex Virus

The C terminus of the herpes simplex virus type 1 origin-binding protein, UL9ct, interacts directly with the viral single-stranded DNA-binding protein ICP8. We show that a 60-amino acid C-terminal deletion mutant of ICP8 (ICP8ΔC) also binds very strongly to UL9ct. Using small angle x-ray scattering, the low resolution solution structures of UL9ct alone, in complex with ICP8ΔC, and in complex with a 15-mer double-stranded DNA containing Box I of the origin of replication are described. Size exclusion chromatography, analytical ultracentrifugation, and electrophoretic mobility shift assays, backed up by isothermal titration calorimetry measurements, are used to show that the stoichiometry of the UL9ct-dsDNA15-mer complex is 2:1 at micromolar protein concentrations. The reaction occurs in two steps with initial binding of UL9ct to DNA (Kd ∼ 6 nm) followed by a second binding event (Kd ∼ 0.8 nm). It is also shown that the stoichiometry of the ternary UL9ct-ICP8ΔC-dsDNA15-mer complex is 2:1:1, at the concentrations used in the different assays. Electron microscopy indicates that the complex assembled on the extended origin, oriS, rather than Box I alone, is much larger. The results are consistent with a simple model whereby a conformational switch of the UL9 DNA-binding domain upon binding to Box I allows the recruitment of a UL9-ICP8 complex by interaction between the UL9 DNA-binding domains.

scholarly journals Ndd, the Bacteriophage T4 Protein That Disrupts theEscherichia coli Nucleoid, Has a DNA Binding Activity

1998 ◽  
Vol 180 (19) ◽  
pp. 5227-5230 ◽  
Author(s):  
Jean-Yves Bouet ◽  
Henry M. Krisch ◽  
Jean-Michel Louarn
Keyword(s):  
Dna Binding ◽  
Bacteriophage T4 ◽  
Binding Activity ◽  
Bacterial Dna ◽  
E Coli ◽  
Dna Binding Activity ◽  
Double Stranded Dna ◽  
Rapid Destruction ◽  
Chromosomal Sequences

ABSTRACT Early in a bacteriophage T4 infection, the phage nddgene causes the rapid destruction of the structure of theEscherichia coli nucleoid. Even at very low levels, the Ndd protein is extremely toxic to cells. In uninfected E. coli, overexpression of the cloned ndd gene induces disruption of the nucleoid that is indistinguishable from that observed after T4 infection. A preliminary characterization of this protein indicates that it has a double-stranded DNA binding activity with a preference for bacterial DNA rather than phage T4 DNA. The targets of Ndd action may be the chromosomal sequences that determine the structure of the nucleoid.

scholarly journals Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43)

2018 ◽  
Vol 293 (41) ◽  
pp. 16083-16099 ◽  
Author(s):  
Friederike Hans ◽  
Marita Eckert ◽  
Felix von Zweydorf ◽  
Christian Johannes Gloeckner ◽  
Philipp J. Kahle
Keyword(s):  
Dna Binding ◽  
Rna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Full Length ◽  
Nuclear Localization Sequence ◽  
Lysine Residues ◽  
Localization Sequence ◽  
Mutagenesis Study

TAR DNA-binding protein of 43 kDa (TDP-43) forms pathological aggregates in neurodegenerative diseases, particularly in certain forms of frontotemporal dementia and amyotrophic lateral sclerosis. Pathological modifications of TDP-43 include proteolytic fragmentation, phosphorylation, and ubiquitinylation. A pathognomonic TDP-43 C-terminal fragment (CTF) spanning amino acids 193–414 contains only four lysine residues that could be potentially ubiquitinylated. Here, serial mutagenesis of these four lysines to arginine revealed that not a single residue is responsible for the ubiquitinylation of mCherry-tagged CTF. Removal of all four lysines was necessary to suppress ubiquitinylation. Interestingly, Lys-408 substitution enhanced the pathological phosphorylation of the immediately adjacent serine residues 409/410 in the context of mCherry-CTF. Thus, Lys-408 ubiquitinylation appears to hinder Ser-409/410 phosphorylation in TDP-43 CTF. However, we did not observe the same effect for full-length TDP-43. We extended the mutagenesis study to full-length TDP-43 and performed MS. Ubiquitinylated lysine residues were identified in the nuclear localization sequence (NLS; Lys-84 and Lys-95) and RNA-binding region (mostly Lys-160, Lys-181, and Lys-263). Mutagenesis of Lys-84 confirmed its importance as the major determinant for nuclear import, whereas Lys-95 mutagenesis did not significantly affect TDP-43's nucleo-cytoplasmic distribution, solubility, aggregation, and RNA-processing activities. Nevertheless, the K95A mutant had significantly reduced Ser-409/410 phosphorylation, emphasizing the suspected interplay between TDP-43 ubiquitinylation and phosphorylation. Collectively, our analysis of TDP-43 ubiquitinylation sites indicates that the NLS residues Lys-84 and Lys-95 have more prominent roles in TDP-43 function than the more C-terminal lysines and suggests a link between specific ubiquitinylation events and pathological TDP-43 phosphorylation.

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