scholarly journals A ribonucleotide reductase from Clostridium botulinum reveals distinct evolutionary pathways to regulation via the overall activity site

2020 ◽  
Vol 295 (46) ◽  
pp. 15576-15587
Author(s):  
Markel Martínez-Carranza ◽  
Venkateswara Rao Jonna ◽  
Daniel Lundin ◽  
Margareta Sahlin ◽  
Lars-Anders Carlson ◽  
...  
Keyword(s):  
Ribonucleotide Reductase ◽  
Clostridium Botulinum ◽  
Building Blocks ◽  
Size Exclusion ◽  
Class I ◽  
Activity Regulation ◽  
E Coli ◽  
X Ray Crystallography ◽  
Exclusion Chromatography ◽  
Insight Into

Ribonucleotide reductase (RNR) is a central enzyme for the synthesis of DNA building blocks. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1–R2 octamers in Escherichia coli. To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size-exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, EM, X-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1–R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1–R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.

scholarly journals PHF13 is a molecular reader and transcriptional co-regulator of H3K4me2/3

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Ho-Ryun Chung ◽  
Chao Xu ◽  
Alisa Fuchs ◽  
Andreas Mund ◽  
Martin Lange ◽  
...  
Keyword(s):  
Dna Damage Response ◽  
Size Exclusion ◽  
X Ray ◽  
Chip Sequencing ◽  
X Ray Crystallography ◽  
Genome Wide ◽  
Damage Response ◽  
Exclusion Chromatography ◽  
Insight Into ◽  
Binding Cell

PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes.

scholarly journals Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miriam F. Suzuki ◽  
Larissa A. Almeida ◽  
Stephanie A. Pomin ◽  
Felipe D. Silva ◽  
Renan P. Freire ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
High Performance ◽  
Signal Sequence ◽  
Osmotic Shock ◽  
Size Exclusion ◽  
Specific Expression ◽  
E Coli ◽  
Human Prolactin ◽  
Exclusion Chromatography

AbstractThe human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 μg/mL/A600 at a final optical density of 3.43 ± 0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF–MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.

scholarly journals Characterization of trimethoprim resistant E. coli dihydrofolate reductase mutants by mass spectrometry and inhibition by propargyl-linked antifolates

2017 ◽  
Vol 8 (5) ◽  
pp. 4062-4072 ◽  
Author(s):  
Michael Cammarata ◽  
Ross Thyer ◽  
Michael Lombardo ◽  
Amy Anderson ◽  
Dennis Wright ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Size Exclusion Chromatography ◽  
Dihydrofolate Reductase ◽  
Native Mass Spectrometry ◽  
Size Exclusion ◽  
E Coli ◽  
Exclusion Chromatography

Native mass spectrometry, size exclusion chromatography, and kinetic assays were employed to study trimethoprim resistance in E. coli caused by mutations P21L and W30R of dihydrofolate reductase.

scholarly journals Characterization of Sorbitol Dehydrogenase SmoS fromSinorhizobium meliloti1021

2019 ◽  
Author(s):  
MacLean G. Kohlmeier ◽  
Ben A. Bailey-Elkin ◽  
Brian L. Mark ◽  
Ivan J. Oresnik
Keyword(s):  
Sinorhizobium Meliloti ◽  
Model Organism ◽  
Time Frame ◽  
Industrial Applications ◽  
Size Exclusion ◽  
Ligand Docking ◽  
X Ray Diffraction ◽  
E Coli ◽  
Exclusion Chromatography

AbstractSinorhizobium meliloti1021 is a Gram-negative alphaproteobacterium with a robust capacity for carbohydrate metabolism. The enzymes that facilitate these reactions assist in the survival of the bacterium across a range of environmental niches, and they may also be suitable for use in industrial processes. SmoS is a dehydrogenase that catalyzes the oxidation of the commonly occurring sugar alcohols sorbitol and galactitol into fructose and tagatose respectively using NAD+as a cofactor. The main objective of this study is to evaluate SmoS using biochemical techniques. The nucleotide sequence was codon optimized for heterologous expression inE. coliBL21 (DE3) GOLD cells, the protein was subsequently overexpressed and purified. Size exclusion chromatography and X-ray diffraction experiments suggest that SmoS is a tetrameric peptide. SmoS was crystallized to 2.1 Å in the absence of substrate and 2.0 Å in complex with sorbitol. SmoS was characterized kinetically and shown to have a preference for sorbitol despite a higher affinity for galactitol. Computational ligand docking experiments suggest that galactitol oxidation proceeds slowly because tagatose binds the protein in a more energetically favorable complex than fructose, and is retained in the active site for a longer time frame following oxidation which reduces the rate of the reaction. These results supplement the inventory of biomolecules with the potential for industrial applications and enhance our understanding of metabolism in the model organismS. meliloti.

scholarly journals Mode of induction of platelet-derived extracellular vesicles is a critical determinant of their phenotype and function

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
P. M. Ferreira ◽  
E. Bozbas ◽  
S. D. Tannetta ◽  
N. Alroqaiba ◽  
R. Zhou ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Size Exclusion ◽  
Biological Functions ◽  
Critical Determinant ◽  
Agonist Stimulation ◽  
Exclusion Chromatography ◽  
Average Size ◽  
And Function ◽  
Free Plasma ◽  
Insight Into

Abstract Platelet-derived extracellular vesicles (PDEVs) are the most abundant amongst all types of EVs in the circulation. However, the mechanisms leading to PDEVs release, their role in coagulation and phenotypic composition are poorly understood. PDEVs from washed platelets were generated using different stimuli and were characterised using nanoparticle tracking analysis. Procoagulant properties were evaluated by fluorescence flow cytometry and calibrated automated thrombography. EVs from plasma were isolated and concentrated using a novel protocol involving a combination of size exclusion chromatography and differential centrifugation, which produces pure and concentrated EVs. Agonist stimulation enhanced PDEV release, but did not alter the average size of EVs compared to those produced by unstimulated platelets. Agonist stimulation led to lower negatively-charged phospholipid externalization in PDEVs, which was reflected in the lower procoagulant activity compared to those generated without agonist stimulation. Circulating EVs did not have externalized negatively-charged phospholipids. None of the 4 types of EVs presented tissue factor. The mechanism by which PDEV formation is induced is a critical determinant of its phenotype and function. Importantly, we have developed methods to obtain clean, concentrated and functional EVs derived from platelet-free plasma and washed platelets, which can be used to provide novel insight into their biological functions.

scholarly journals A Supramolecular Assembly of Hemoproteins Formed in a Star-Shaped Structure via Heme–Heme Pocket Interactions

2021 ◽  
Vol 22 (3) ◽  
pp. 1012
Author(s):  
Julian Wong Soon ◽  
Koji Oohora ◽  
Shota Hirayama ◽  
Takashi Hayashi
Keyword(s):  
Supramolecular Assembly ◽  
Building Blocks ◽  
Size Exclusion ◽  
Hydrodynamic Diameter ◽  
Prosthetic Group ◽  
Heme Pocket ◽  
Cytochrome B562 ◽  
Sds Page ◽  
Vis Spectroscopy ◽  
Exclusion Chromatography

Proteins have been used as building blocks to provide various supramolecular structures in efforts to develop nano-biomaterials possessing broad biological functionalities. A series of unique structures have been obtained from the engineering of hemoproteins which contain the iron porphyrin known as heme, as a prosthetic group. This work in developing assembling systems is extended using cytochrome b562, a small electron transfer hemoprotein engineered to include an externally-attached heme moiety. The engineered units, which form a one-dimensional assembly via interprotein heme–heme pocket interactions, are conjugated to an apo-form of hexameric tyrosine-coordinated hemoprotein (apoHTHP) to provide a branching unit promoting the assembly of a star-shaped structure. The incorporation of the heme moiety attached to the protein surface of cytochrome b562 into apoHTHP can be accelerated by elevating the reaction temperature to generate a new assembly. The formation of a new larger assembly structure was confirmed by size exclusion chromatography. The ratio of the heme-containing units in the assemblies was analyzed by UV-Vis spectroscopy and the population of protein units estimated from SDS PAGE suggests the presence of plausible star-shaped structures, which are supported by hydrodynamic diameter data obtained by dynamic light scattering.

scholarly journals Use of photoimmunoconjugates to characterize ABCB1 in cancer cells

Nanophotonics ◽  
2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Barry J. Liang ◽  
Sabrina Lusvarghi ◽  
Suresh V. Ambudkar ◽  
Huang-Chiao Huang
Keyword(s):  
Cancer Cells ◽  
Triple Negative ◽  
Size Exclusion ◽  
Detection Methods ◽  
Drug Resistant ◽  
Drug Transporter ◽  
Open Conformation ◽  
Exclusion Chromatography ◽  
Insight Into

Abstract Accurate detection of ATP-binding cassette drug transporter ABCB1 expression is imperative for precise identification of drug-resistant tumors. Existing detection methods fail to provide the necessary molecular details regarding the functional state of the transporter. Photoimmunoconjugates are a unique class of antibody–dye conjugates for molecular diagnosis and therapeutic treatment. However, conjugating hydrophobic photosensitizers to hydrophilic antibodies is quite challenging. Here, we devise a photoimmunoconjugate that combines a clinically approved benzoporphyrin derivative (BPD) photosensitizer and the conformational-sensitive UIC2 monoclonal antibody to target functionally active human ABCB1 (i.e., ABCB1 in the inward-open conformation). We show that PEGylation of UIC2 enhances the BPD conjugation efficiency and reduces the amount of non-covalently conjugated BPD molecules by 17%. Size exclusion chromatography effectively separates the different molecular weight species found in the UIC2–BPD sample. The binding of UIC2–BPD to ABCB1 was demonstrated in lipidic nanodiscs and ABCB1-overexpressing triple negative breast cancer (TNBC) cells. UIC2–BPD was found to retain the conformation sensitivity of UIC2, as the addition of ABCB1 modulators increases the antibody reactivity in vitro. Thus, the inherent fluorescence capability of BPD can be used to label ABCB1-overexpressing TNBC cells using UIC2–BPD. Our findings provide insight into conjugation of hydrophobic photosensitizers to conformation-sensitive antibodies to target proteins expressed on the surface of cancer cells.

scholarly journals Structural insight into the dual function of LbpB in mediating Neisserial pathogenesis

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Ravi Yadav ◽  
Srinivas Govindan ◽  
Courtney Daczkowski ◽  
Andrew Mesecar ◽  
Srinivas Chakravarthy ◽  
...  
Keyword(s):  
Antimicrobial Peptide ◽  
Iron Acquisition ◽  
Bound State ◽  
Size Exclusion ◽  
Dual Function ◽  
Binding Studies ◽  
Cationic Antimicrobial Peptide ◽  
X Ray Scattering ◽  
Exclusion Chromatography ◽  
Insight Into

Lactoferrin-binding protein B (LbpB) is a lipoprotein present on the surface of Neisseria that has been postulated to serve dual functions during pathogenesis in both iron acquisition from lactoferrin (Lf), and in providing protection against the cationic antimicrobial peptide lactoferricin (Lfcn). While previous studies support a dual role for LbpB, exactly how these ligands interact with LbpB has remained unknown. Here, we present the structures of LbpB from N. meningitidis and N. gonorrhoeae in complex with human holo-Lf, forming a 1:1 complex and confirmed by size-exclusion chromatography small-angle X-ray scattering. LbpB consists of N- and C-lobes with the N-lobe interacting extensively with the C-lobe of Lf. Our structures provide insight into LbpB’s preference towards holo-Lf, and our mutagenesis and binding studies show that Lf and Lfcn bind independently. Our studies provide the molecular details for how LbpB serves to capture and preserve Lf in an iron-bound state for delivery to the membrane transporter LbpA for iron piracy, and as an antimicrobial peptide sink to evade host immune defenses.

scholarly journals Serendipitous crystallization of E. coli HPII catalase, a sequel to “the tale usually not told”

2021 ◽  
Author(s):  
Marta Grzechowiak ◽  
Bartosz Sekula ◽  
Mariusz Jaskolski ◽  
Milosz Ruszkowski
Keyword(s):  
Molecular Weight ◽  
Carbonic Anhydrase ◽  
Recombinant Protein ◽  
Metal Binding ◽  
Recombinant Protein Expression ◽  
Crystal Form ◽  
Size Exclusion ◽  
E Coli ◽  
Low Concentrations ◽  
Exclusion Chromatography

Protein crystallographers are well aware of the trap of crystallizing E. coli proteins instead of the macromolecule of interest if heterologous recombinant protein expression in E. coli was part of the experimental pipeline. Among the well-known culprits are YodA metal-binding lipocalin (25 kDa) and YadF carbonic anhydrase (a tetramer of 25 kDa subunits). We report a novel crystal form of another such culprit, E. coli HPII catalase, which is a tetrameric protein of ~340 kDa molecular weight. HPII is likely to contaminate recombinant protein samples, co-purify, and then co-crystallize with the target proteins, especially if their masses in size exclusion chromatography are ~300–400 kDa. What makes this case more interesting but also parlous, is the fact that HPII can crystallize from very low concentrations, even well below 1 mg/mL.

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