17 research outputs found
Surface properties of CtDsbA.
<p>Surface representation for CtDsbA of the catalytic (left) and non-catalytic (right) faces. The active site residues Cys-Ser-Ala-Cys are colored yellow and the nucleophilic cysteine sulfur highlighted in orange. Pockets formed on the posterior face of the protein between H1 and H3 (pocket 1) and the N-terminal unstructured region and H6 (pocket 2) are labeled. B. Electrostatic surface representation of CtDsbA. Views are oriented as above. Electrostatic surface potential is contoured between -5 (red) and +5 (blue) kT/e. The nucleophilic cysteine is annotated with an S.</p
Crystal structure of CtDsbA.
<p>A. The crystal structure of CtDsbA contains a thioredoxin domain (light green) and a helical domain (dark green.) Loops on the catalytic surface that constitute the active site of CtDsbA and determine redox activity are colored orange and labeled. The active site catalytic disulfide is highlighted with sulfurs shown as yellow spheres. The non-catalytic disulfide (between Cys84 and Cys145) and the single thiol (Cys70) in L1 are shown in stick representation. The most N-terminal region of CtDsbA is unstructured. Crystal packing interactions with the second monomer in the asymmetric unit and a symmetry related molecule (shown in white) stabilize this region of the protein such that is well resolved in the electron density map. B. Close view of the four loops (L1, <i>cis</i>Pro L2, L3 and the Cys-Ser-Ala-Cys motif) which constitute the active site surface of CtDsbA. C. In the crystal structure the active site cysteines are oxidized. Analysis of bond distances indicates that the Cys 38 thiolate could be stabilized by favorable bond interactions with Thr 172 (3.4 Å between Thr 172 OH and Cys 38 SG in the oxidized structure) of the neighboring cisPro L2 consistent with an oxidizing protein character. The Cys 41 sulfur atom is 3.5 Å from the Thr 172 hydroxyl in the oxidized structure. 2Fo-Fc and Fo-Fc electron density maps for the active site and cisPro Loop 2 were generated from calculated phases using phenix.maps and are shown contoured at 1.0 σ and 3.0 σ respectively. The maps are shown within a 1 Å radius of each atom of each loop.</p
Biochemical characterization of CtDsbA.
<p><b>A</b> Reduction of insulin (131 μM) was measured as increase in absorbance at 650nm in 0.1mM sodium phosphate buffer, pH 7, 2mM EDTA. The reaction was performed in the absence (■) or presence of 10 μM EcDsbC (●), EcDsbA (♦) or CtDsbA (○). Representative data are shown for the absence and presence of 10 μM EcDsbA. Mean and SD are shown for two biological replicates (three biological replicates for CtDsbA). <b>B</b> 80 nM EcDsbA (<b>▼</b>) and 320 nM CtDsbA (●), MtbDsbA (■) and WpDsbA1 (<b>▲)</b> oxidize a fluorescently labeled protein in the presence of 2 mM GSSG. GSSG shows only limited oxidizing activity in the absence of a DsbA protein (■). The buffer only control (○) shows no oxidizing activity. For MtbDsbA, WpDsbA1, EcDsbA and CtDSbA mean and SD of two biological replicates are shown (for each biological replicates four technical replicates was performed.) For the buffer and GSSG only controls, mean and sd for four technical replicates are shown. <b>C</b> Isomerase activity was assessed as the ability to refold scrambled RNAseA. ScRNase (40 μM) was incubated in 0.1 M sodium phosphate buffer pH 7.0, 1 mM EDTA, 10 μM DTT in the absence and presence of 10 μM EcDsbA (■), EcDsbC (○) and CtDsbA (●). RNase activity was monitored as the cleavage of cCMP which leads to an increase in absorbance at 296 nm. Mean and SD for three biological replicates is shown for CtDsbA. EcDsbC and EcDsbA is able to restore ~80% and ~20% of RNaseA activity, which is equivalent to what has been reported previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168485#pone.0168485.ref008" target="_blank">8</a>] <b>D</b> Temperature induced unfolding of oxidized (○) and reduced (●) CtDsbA was determined by far UV CD spectroscopy. The thermal unfolding of CtDsbA results in an increase in molar ellipticity at 220 nm showing that the reduced form of CtDsbA is more stable than the oxidized form. Mean and SD are shown for two biological replicates.</p
Sequence alignment of DsbAs with two disulfide bonds.
<p>Sequence alignment of structurally characterised DsbAs with two disulfide bonds. Sequences were aligned using Clustal Omega and visualized using ESPript 3.0 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168485#pone.0168485.ref053" target="_blank">53</a>]. Secondary structural elements are shown for the structure of CtDsbA. Disulfide bonds are indicated with black connecting lines and labeled S-S. The single unpaired cysteine C70 is highlighted in yellow.</p
X-ray data measurement and refinement statistics for CtDsbA.
<p>X-ray data measurement and refinement statistics for CtDsbA.</p
Redox potential determination for CtDsbA-SSS by electrophoretic motility shift.
<p><b>A</b> SDS-PAGE gel of oxidized CtDsbA (3 μM) incubated for 24 h with increasing concentration of DTT (0 μM -12 mM). <b>B</b> The fraction of thiolate as a function of reduced DTT versus oxidized DTT is plotted. Fitting of the data revealed a Keq of 3.8 ± 0.8 x 10<sup>−4</sup> M equivalent to a redox potential of -229 mV. Mean and SD calculated from 4 biological replicates are plotted.</p
CtDsbA does not complement EcDsbA.
<p><i>ecdsbA</i> null <i>E</i>. <i>coli</i> are unable to swarm on soft agar. Exogenous expression of EcDsbA under an arabinose inducible promoter is able to complement this non-motile phenotype as shown by the swarming halo around the original inoculation point (upper panel, EcDsbA induced). CtDsbA is not able to restore the phenotype. Neither EcDsbA nor CtDsbA are able to restore mobility in a <i>ecdsbA/ecdsbB</i> double null <i>E</i>. <i>coli</i> strain (lower panels) indicating the requirement for EcDsbB to maintain a pool of oxidized DsbA.</p
Control isolates used during development of <i>Ct</i>GEM typing.
<p>Control isolates used during development of <i>Ct</i>GEM typing.</p
Numbers and percentages of successful PCR amplifications from 113 <i>C</i>. <i>trachomatis</i> positive clinical specimens.
<p>Numbers and percentages of successful PCR amplifications from 113 <i>C</i>. <i>trachomatis</i> positive clinical specimens.</p
HRMA analysis of the <i>ompA</i> fragment (ofr).
<p>(A) Sequence alignment of the known haplotypes of ofr showing the primer sequences (grey shading). (B) HRMA curves showing the resolution of the genotypes. A detailed guide for the HRMA is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195454#pone.0195454.s002" target="_blank">S2 File</a> in the supplemental material.</p