Structural features of the NifL PAS1 domain and its flanking regions.

<p>(A) Summary of NifL fragments used to analyse the influence of flanking regions on the properties of the PAS1 domain, indicated below the domain architecture of the complete protein. The oligomerisation state of each fragment, as determined by size exclusion chromatography in this work, is indicated in brackets. (B) The predicted secondary structure of NifL residues 1–160 as determined by the PSIPRED server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046651#pone.0046651-Buchan1" target="_blank">[18]</a> is shown above the amino acid sequence. Amino acids resolved in the crystal structure of the oxidized form of PAS1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046651#pone.0046651-Key1" target="_blank">[13]</a> are underlined in black. The secondary structure plot for PDB code 2gj3: chain A (from PDBsum <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046651#pone.0046651-Laskowski1" target="_blank">[30]</a>) is displayed above the predicted structure. N-terminal and C-terminal flanking sequences are underlined in green and red respectively.</p

Influence of N-terminal residues on PAS1 oligomerisation state in the context of the 1–140 construct.

<p>(A) SEC analysis of wild-type and truncated variants (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046651#pone-0046651-g003" target="_blank">Figure 3</a> for N-terminal sequences). In each case, the hexa-histidine-containing tag encoded by the pETNdeM-11 vector provides 26 additional residues at the N-terminus. SEC was performed at a protein concentration of 106 µM (expressed in monomeric terms). (B) The association state determined in (A) for each protein is plotted against the number of residues removed from the N-terminus.</p

Influence of N-terminal truncations on the ability of NifL to regulate transcriptional activation by NifA.

<p>The number of residues missing from the N-terminus (including the initial methionine) is indicated numerically on the x axis and the sequence of each truncation is represented below. “R" indicates a control in which only the reporter plasmid was present. In all other cases, <i>nifA</i> was present on the plasmid together with <i>nifL</i> or its variants. “A" indicates the plasmid encodes an in-frame deletion, containing 65 C-terminal residues of NifL (pPR39). This plasmid acts as a control for NifA activity in the absence of active NifL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046651#pone.0046651-Sderbck1" target="_blank">[14]</a>. “wt" indicates native NifL. Cultures were assayed for β-galactosidase activity as a reporter of NifA-mediated transcriptional activation from the <i>nifH_p-lacZ</i> fusion on plasmid pRT22 as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046651#pone.0046651-Slavny1" target="_blank">[12]</a>. Cultures were grown under the following conditions; anaerobically under nitrogen limitation with casein hydrolysate as the sole nitrogen source (black bars), anaerobically with (NH₄)₂SO₄ as nitrogen source (grey bars), and aerobically with casein hydrolysate as sole nitrogen source (white bars). (Assays were also performed on cultures grown anaerobically in the presence of (NH₄)₂SO₄, but β-galactosidase activities were too low to display on this scale.) All experiments were performed at least in duplicate with error bars denoting the standard error of the mean.</p

Influence of deletions in the α-helical linker on the ability of NifL to inhibit NifA plotted as the change in helix angle.

<p>The data is derived from the results in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046651#pone-0046651-g004" target="_blank">Figure 4A</a>. Cultures were grown with casein hydrolysate as the sole nitrogen source, either under aerobic (solid circles, solid line) or anaerobic conditions (open circles, dotted line).</p