Molecular weights of GyrA-CTD proteins calculated from their amino acid sequences and elution volumes from analytical size exclusion chromatography.

<p>Molecular weights of GyrA-CTD proteins calculated from their amino acid sequences and elution volumes from analytical size exclusion chromatography.</p

Proportion of secondary structures calculated from CD-spectra using the Dichroweb server [32].

<p>Proportion of secondary structures calculated from CD-spectra using the Dichroweb server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.ref032" target="_blank">32</a>].</p

Analyses of the MBP-PfGyrA-CTD variant without the Asn-rich region.

<p>MBP-PfGyrA-CTD without the Asn-rich region (887–902) was subjected to <b>A.</b> EMSA in the presence of 1 nM substrate 140 bp DNA and the indicated excess of protein and <b>B</b>. topology footprinting assay performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.g005" target="_blank">Fig 5</a>. MBP-PfGyrA-CTD(ΔN) exhibits similar affinity for DNA when compared to MBP-PfGyrA-CTD. No difference could be observed in terms of ability to wrap DNA.</p

DNA binding by GyrA-CTD proteins demonstrated by EMSA and fluorescence anisotropy assays.

<p><b>A</b> EcGyrA-CTD(ΔT) binds DNA as previously reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.ref034" target="_blank">34</a>]. MBP-PfGyrA-CTD also binds DNA, apparently with higher affinity. <b>B</b> The experiment repeated with PfGyrA-CTD fragment without a tag. This CTD also exhibits a higher affinity for DNA. For A and B, protein concentration in terms of multiples of DNA concentration is given above each lane. <b>C</b> DNA binding by GyrA-CTD tested by fluorescence anisotropy. Fluorescence anisotropy by FAM-labelled DNA at 50 nM was measured as a function of protein concentration from 0 to 3200 nM. Error bars indicate standard deviations from three independent measurements. The presence and the absence of the acidic C-terminal tail (854–875) affects the DNA binding of EcGyrA-CTD negatively and positively, respectively, as previously reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.ref031" target="_blank">31</a>] and were employed as control proteins in this assay. Bmax and Kd respectively for each protein are as follows. EcGyrA-CTD(ΔT), 94.28 mA ± 15.63 mA, 1114.60 nM ± 430.53 nM; MBP-PfGyrA-CTD, 194.32 mA ± 7.98 mA, 205.49 nM ± 31.40 nM; MBP-PfGyrA-CTD(ΔN), 189.82 mA ± 11.62 mA, 221.55 nM ± 49.50 nM. Values were not calculated for EcGyrA-CTD(WT).</p

Topology footprinting assay for GyrA-CTD proteins.

<p>Ability of GyrA-CTD proteins to wrap DNA was visualised by incubating approx. 2.7 nM nicked (topologically free) DNA with the indicated molar excess of protein; subsequently the nick was sealed by T4 ligase, allowing the topological changes introduced as a result of protein-DNA interaction to be observed. EcGyrA-CTD(ΔT) and EcGyrA-CTD(WT) behaved as previously reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142313#pone.0142313.ref031" target="_blank">31</a>]. Similar to EcGyrA-CTD(ΔT), MBP-PfGyrA-CTD and PfGyrA-CTD introduced writhe into topologically free nicked plasmid DNA, and the effect increases in a concentration dependent manner. “R” and “S” represent the positions of relaxed and supercoiled DNA respectively and apply to all gels in the figure.</p

Purification of QL-nanoKAZ from 800 mL of cultured <i>E</i>. <i>coli</i> cells using a Ni-chelate column.

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Expression of QL-nanoKAZ in the presence or absence of the secretory signal peptide sequence from <i>Gaussia</i> luciferase (GLsp) in CHO-K1 cells.

Expression of QL-nanoKAZ in the presence or absence of the secretory signal peptide sequence from Gaussia luciferase (GLsp) in CHO-K1 cells.</p

Luminescence properties of QL-nanoKAZ.

A. Luminescence kinetics of QL-nanoKAZ with CTZ and its analogs as substrates. B. Normalized luminescence spectra of QL-nanoKAZ with CTZ and its analogs, based on the luminescence intensity of QL-nanoKAZ with CTZ. C. Linearity of luminescence intensity (Imax) of QL-nanoKAZ with CTZ, in comparison with nanoKAZ, SNH-nanoKAZ, GLase, and aequorin at the protein concentrations of 0.3 pg to 3 ng (n = 6). Solid and dashed lines represent blank + 3 SD for aequorin and the CTZ-utilizing luciferases, respectively.</p