Journal: Nucleic Acids Research

Article Title: Insight into F plasmid DNA segregation revealed by structures of SopB and SopB–DNA complexes

doi: 10.1093/nar/gkq161

Figure Lengend Snippet: Selected crystallographic data for SopB–DNA structures

Article Snippet: An artificial sopB gene, encoding the full-length (FL) SopB protein, was codon optimized for Escherichia coli expression and purchased from Genscript Corporation, Piscatway, NJ, USA ( www.genscript.com ).

Techniques:

Journal: Nucleic Acids Research

Article Title: Insight into F plasmid DNA segregation revealed by structures of SopB and SopB–DNA complexes

doi: 10.1093/nar/gkq161

Figure Lengend Snippet: Selected crystallographic data for SopB(275–323)

Article Snippet: An artificial sopB gene, encoding the full-length (FL) SopB protein, was codon optimized for Escherichia coli expression and purchased from Genscript Corporation, Piscatway, NJ, USA ( www.genscript.com ).

Techniques:

Journal: Nucleic Acids Research

Article Title: Insight into F plasmid DNA segregation revealed by structures of SopB and SopB–DNA complexes

doi: 10.1093/nar/gkq161

Figure Lengend Snippet: Crystal structures of SopB-18mer complexes. ( A ) A section of the experimental MAD electron density map (shown as a blue mesh) for the FL SopB-18mer complex, contoured at 1.5σ and calculated to 3.5 Å resolution. Labeled are the location of the pseudocontinuous DNA and one of the SopB intermolecular dimers, which bridge between DNA duplexes to form the crystal lattice. Also labeled is one of the large solvent channels. ( B ) Ribbon diagram of the I23 SopB-18mer complex. The crystallographic asymmetric unit (ASU) consists of two subunits (cyan and magenta) and one 18-mer DNA duplex. Shown also are subunits involved in secondary dimer/bridging interactions, generated in the crystals. For the cyan subunit, the secondary structural elements are labeled and the first and last residues observed in the structure are labeled N and C, respectively. This figure ( C and D ) and Figures 2A–D, 3, 4, 5A and C, and 6A and B were made with PyMOL (55). (C) Ribbon diagram of the P3 1 21 SopB(155–272)-18mer complex. The molecules in the ASU are all shown consisting of three SopB secondary dimers and four 18mer duplexes, which all pack pseudocontinuously in the crystal. The specific HTH-major groove interacting subunits are labeled canonical major groove and the one subunit that interacts non-specifically with two minor grooves (colored blue) is also shown and its minor groove contacts labeled. (D) Ribbon diagram of the P2 1 SopB(155–272)-18mer complex. The ASU consists of one secondary dimer and two SopB subunits and two 18-mer DNA duplexes.

Article Snippet: An artificial sopB gene, encoding the full-length (FL) SopB protein, was codon optimized for Escherichia coli expression and purchased from Genscript Corporation, Piscatway, NJ, USA ( www.genscript.com ).

Techniques: Labeling, Solvent, Generated

Journal: Nucleic Acids Research

Article Title: Insight into F plasmid DNA segregation revealed by structures of SopB and SopB–DNA complexes

doi: 10.1093/nar/gkq161

Figure Lengend Snippet: Structure of SopB(275–323) dimer-domain. ( A ) Left, a section of the experimental MIR electron density map for the SopB(275–323) structure (blue mesh) contoured at 1σ. Right, ribbon diagram of the SopB(275–323) structure with one subunit colored red and labeled, and the other subunit colored green. ( B ) Comparison of the P1 ParB dimer-domain–DNA complex (right) with the corresponding SopB dimer-domain (left). Note that although the overall structure and topology are similar, P1 ParB has extended loops between its β1–β2 and β2–β3 units that are responsible for DNA binding that are not present in SopB. ( C ) Comparison of the electrostatic surfaces of the P1 ParB and SopB. Shown is the helical containing face of each dimer-domain, which is strongly electronegative (red) in both structures. ( D ) Electrostatic surface of the face opposite to that shown in (C). This face, which is involved in DNA-binding in the P1 ParB protein, is electropositive (blue) in each structure.

Article Snippet: An artificial sopB gene, encoding the full-length (FL) SopB protein, was codon optimized for Escherichia coli expression and purchased from Genscript Corporation, Piscatway, NJ, USA ( www.genscript.com ).

Techniques: Labeling, Comparison, Binding Assay

Journal: Nucleic Acids Research

Article Title: Insight into F plasmid DNA segregation revealed by structures of SopB and SopB–DNA complexes

doi: 10.1093/nar/gkq161

Figure Lengend Snippet: SopB primary and secondary (bridging) dimers. Relationship of the SopB primary and secondary dimers. The structure of the SopB primary dimer bound to the palindromic DNA site was produced by combining the DNA-binding and dimer-domains (the flexible linkage is indicated by dashed lines). Above is shown the secondary dimer contacts that permit SopB to bridge or spread between multiple, adjacent DNA sites (generated by a 90° rotation).

Article Snippet: An artificial sopB gene, encoding the full-length (FL) SopB protein, was codon optimized for Escherichia coli expression and purchased from Genscript Corporation, Piscatway, NJ, USA ( www.genscript.com ).

Techniques: Produced, Binding Assay, Generated

Journal: Nucleic Acids Research

Article Title: Insight into F plasmid DNA segregation revealed by structures of SopB and SopB–DNA complexes

doi: 10.1093/nar/gkq161

Figure Lengend Snippet: The SopB α6–α7 secondary dimer interactions. ( A ) Ribbon diagram showing the residues that are involved in the formation of the secondary dimer interaction. Two views are included that are related by a ∼90° rotation. Residues that contribute to the interface are shown as sticks and labeled. Also labeled are α6 and α7. ( B ) Superimposition of subunits of all the secondary dimers reveals that the dimerization is not symmetric but mediated by one subunit making one set of contacts and the other making a different set of contacts. This asymmetric arrangement is observed in all dimers.

Article Snippet: An artificial sopB gene, encoding the full-length (FL) SopB protein, was codon optimized for Escherichia coli expression and purchased from Genscript Corporation, Piscatway, NJ, USA ( www.genscript.com ).

Techniques: Labeling

Journal: Nucleic Acids Research

Article Title: Insight into F plasmid DNA segregation revealed by structures of SopB and SopB–DNA complexes

doi: 10.1093/nar/gkq161

Figure Lengend Snippet: SopB–DNA interactions. ( A ) Left, schematic representation of SopB–DNA interactions. Only one half site of the 18-mer duplex is shown as the identical contacts are made to each half site. The strands are labeled 1–9 and 1′–9′ (where ′ indicates other strand of the duplex). Bases are represented as rectangles and labeled according to sequence. The ribose groups are shown as pentagons. Hydrophobic contacts are indicated by lines and hydrogen bonds, by arrows. Right, close up of the SopB–DNA specific major groove interactions that are made to each half site and indicated schematically. Interacting residues are shown as blue sticks and the secondary-structural elements are labeled. ( B ) Fluorescence polarization DNA-binding isotherms comparing SopB binding to the 43- and 18-mer centromere sites. Each data set was normalized, and normalized polarizations were plotted along the y -axis against the protein concentrations, which are plotted along the x -axis. ( C ) Close up of the triple bridging interaction that is comprised of specific and non-specific SopB–DNA found in the P3 1 21 crystal form. Specifically, this dimer is the one that is also colored blue and magenta in C. The blue subunit makes non-specific minor groove contacts to two DNA duplexes while the magenta subunit makes the canonical major groove contacts indicated in A and found in all the subunits of the crystals except the P3 1 21 blue subunit.

Article Snippet: An artificial sopB gene, encoding the full-length (FL) SopB protein, was codon optimized for Escherichia coli expression and purchased from Genscript Corporation, Piscatway, NJ, USA ( www.genscript.com ).

Techniques: Labeling, Sequencing, Fluorescence, Binding Assay

Journal: Nucleic Acids Research

Article Title: Insight into F plasmid DNA segregation revealed by structures of SopB and SopB–DNA complexes

doi: 10.1093/nar/gkq161

Figure Lengend Snippet: Electrostatic surface representations of SopB-18mer specific and non-specific complexes. ( A ) SopB dimers are shown as surface representations with electropositive regions colored blue and electronegative regions red. The DNA is shown as sticks. Shown is the specific complex in which the DNA major grooves are contacted by the basic HTH motifs. ( B ) Electrostatic surface representation of the non-specific SopB–DNA complex. In this complex, the SopB secondary dimer bridges three different DNA duplexes by making non-specific contacts to the minor grooves of two DNA duplexes (from one subunit) and specific contacts to the major groove by the HTH of the second subunit.

Article Snippet: An artificial sopB gene, encoding the full-length (FL) SopB protein, was codon optimized for Escherichia coli expression and purchased from Genscript Corporation, Piscatway, NJ, USA ( www.genscript.com ).

Techniques:

Journal: Frontiers in Plant Science

Article Title: Molecular Analysis of Protein-Protein Interactions in the Ethylene Pathway in the Different Ethylene Receptor Subfamilies

doi: 10.3389/fpls.2019.00726

Figure Lengend Snippet: Expression and purification of recombinant AtETR2. (A) E. coli C43(DE) strain was used for heterologous expression of Arabidopsis thaliana receptor ETR2. Expression was analyzed by SDS-PAGE and immunoblotting. Protein expression was monitored 1 (lane 1) to 5 h (lane 5) after induction with IPTG and detected by an anti-His antibody. AtETR2 migrates on SDS gels with an apparent molecular mass of 120 kDa. (B) His-tagged AtETR2 was purified by IMAC, separated by SDS-PAGE and visualized by colloidal Coomassie staining and (C) immunoblotting using an anti-His antibody.

Article Snippet: Codon optimized cDNA encoding full-length AtETR2 (UniProt ID: Q0WPQ2) was purchased from GenScript USA according to the published sequence (NCBI ID: NM_113216.3).

Techniques: Expressing, Purification, Recombinant, SDS Page, Western Blot, Staining

Journal: Frontiers in Plant Science

Article Title: Molecular Analysis of Protein-Protein Interactions in the Ethylene Pathway in the Different Ethylene Receptor Subfamilies

doi: 10.3389/fpls.2019.00726

Figure Lengend Snippet: Circular dichroism spectra of AtETR2. (A) The far-UV spectra of AtETR2 was calculated and adjusted to molar extinction (∆ɛ) considering molecular weight and protein concentration of AtETR2. (B) Secondary structure content was calculated by CONTINLL (solid line) and CDSSTR (dashed line) from the CDpro software package.

Article Snippet: Codon optimized cDNA encoding full-length AtETR2 (UniProt ID: Q0WPQ2) was purchased from GenScript USA according to the published sequence (NCBI ID: NM_113216.3).

Techniques: Circular Dichroism, Molecular Weight, Protein Concentration, Software

Journal: Frontiers in Plant Science

Article Title: Molecular Analysis of Protein-Protein Interactions in the Ethylene Pathway in the Different Ethylene Receptor Subfamilies

doi: 10.3389/fpls.2019.00726

Figure Lengend Snippet: Autophosphorylation of purified AtETR2 was performed with 0.1 mM [γ- 32 P]ATP and magnesium as cofactor. Proteins were detected by (A) Coomassie staining. (B) Incorporation of 32 P was measured by autoradiography for 6 days. Experiments were performed using AtETR2 solubilized and purified without ATP purification step (1) or chemically and thermally denatured AtETR2 (2).

Article Snippet: Codon optimized cDNA encoding full-length AtETR2 (UniProt ID: Q0WPQ2) was purchased from GenScript USA according to the published sequence (NCBI ID: NM_113216.3).

Techniques: Purification, Staining, Autoradiography

Journal: Frontiers in Plant Science

Article Title: Molecular Analysis of Protein-Protein Interactions in the Ethylene Pathway in the Different Ethylene Receptor Subfamilies

doi: 10.3389/fpls.2019.00726

Figure Lengend Snippet: Interaction studies of Arabidopsis ETR2 and EIN2 by MST. Dissociation constants of the interactions were obtained from the related binding curves. Titration of unlabeled AtEIN2 479-1294 to AtETR2 (●) is described by a dissociation constant ( K d ) of 161(30) nM. Chemically and thermally denatured AtEIN2 479-1294 shows no binding event to AtETR2 (▲). Binding of unlabeled AtETR2 to AtEIN2 479-129 is represented by a K d value of 147(15) nM (○). All data represent the mean (SD) of three independent measurements (●, ○) and duplicates (▲), respectively.

Article Snippet: Codon optimized cDNA encoding full-length AtETR2 (UniProt ID: Q0WPQ2) was purchased from GenScript USA according to the published sequence (NCBI ID: NM_113216.3).

Techniques: Binding Assay, Titration

Journal: Frontiers in Plant Science

Article Title: Molecular Analysis of Protein-Protein Interactions in the Ethylene Pathway in the Different Ethylene Receptor Subfamilies

doi: 10.3389/fpls.2019.00726

Figure Lengend Snippet: MST based protein-protein interaction assay between AtCTR1 and receptor proteins AtETR1 and AtETR2. Binding of AtETR1 to fluorescently labeled AtCTR1 measured by MST resulted in a K d value of 169(15) nM (○). For AtCTR1-AtETR2 complex formation a K d value of 165(20) nM was obtained (●). As negative control, titration of chemically denatured AtCTR1 with AtETR2 is shown. Here, no binding event was observed (▲). Data are given as the mean (SD) of independent triplicates (●, ○) and duplicates (▲), respectively.

Article Snippet: Codon optimized cDNA encoding full-length AtETR2 (UniProt ID: Q0WPQ2) was purchased from GenScript USA according to the published sequence (NCBI ID: NM_113216.3).

Techniques: Protein Protein Interaction Assay, Binding Assay, Labeling, Negative Control, Titration

Journal: Frontiers in Plant Science

Article Title: Molecular Analysis of Protein-Protein Interactions in the Ethylene Pathway in the Different Ethylene Receptor Subfamilies

doi: 10.3389/fpls.2019.00726

Figure Lengend Snippet: Quantification of receptor-receptor interactions by microscale thermophoresis. (A) For the homomeric AtETR1-AtETR1 complex formation a K d value of 326(18) nM (○) was obtained. As negative control chemically denatured AtETR1 was used showing no binding event (△). From the binding curve of the homomeric AtETR2-AtETR2 complex a K d value of 96(18) nM (●) was calculated. Chemically denatured AtETR2 indicates no interaction of the binding partners (▲). All data represent the mean (SD) of independent triplicates (○, ●) and duplicates (△, ▲). (B) Summary of the dissociation constants K d for receptor-receptor interactions obtained by MST, also see . All data represent the mean (SD) of three independent measurements.

Article Snippet: Codon optimized cDNA encoding full-length AtETR2 (UniProt ID: Q0WPQ2) was purchased from GenScript USA according to the published sequence (NCBI ID: NM_113216.3).

Techniques: Microscale Thermophoresis, Negative Control, Binding Assay