10 research outputs found
Ligand-induced changes in the vestibule and grotto region of luciferase.
<p>The “active” DSLA bound (left, 2D1S.pdb) and ATP bound (right, 2D1Q.pdb) structures are compared from the same viewing points. Upper panels show a cross section through the vestibule and grotto region beyond the luciferin binding pocket (compare <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#pone-0029854-g007" target="_blank">Fig. 7</a>). The lower panels show the vestibule as viewed from the luciferin pocket; the left panel shows the O10 of DLSA for orientation. Water molecules are shown in red in the +ATP structure and in gold in the DSLA structure (one of the eight water molecules is partially hidden in panel C and is indicated with a circle). The cross-section surface capping is semitransparent. Surface coloring is grey for carbon, red for oxygen and blue for nitrogen. DSLA (gold carbons in A) is a surrogate for luciferin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#pone-0029854-g001" target="_blank">Fig. 1B</a>). Similar changes are seen in American luciferase (3IES.pdb and 3IEP.pdb).</p
Relationship of photolabeled residues to other ligands bound.
<p>The structure of American luciferase with two bromoform molecules bound (1BA3.pdb; bromine atoms are brown) was superimposed on the DSLA–bound Japanese luciferase structure (2D1S.pdb). A cross section of 2D1S is shown without surface capping in a surfaced ribbon diagram (light blue) with the photolabeled residues shown with cyan carbons. DLSA is shown with gold carbons. The surface of the bromoforms is shown in mesh representation. The carbon atom of the inner bromoform (on the right), is centered within 2 Å of the terminal oxygen (O10) of DSLA and overlaps C1, C5 and C6 of the benzothiazole ring of DSLA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#pone-0029854-g007" target="_blank">Fig. 7</a> for numbering). The outer bromoform (left) is situated in the grotto region 2.7 Å from Glu-313. It clashes sterically with the superimposed Japanese luciferase structure.</p
Identification of photolabeled residues in the peptide Gln-340 – Lys-366.
<p>Conditions are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#pone-0029854-g004" target="_blank">Fig. 4</a>. <b>A.</b> 3-azibutanol (1 mM) photolabels Asp-358. <b>B.</b> TFD-benzyl alcohol (100 µM) photolabels Ser-349.</p
The structure of Japanese luciferase in the vicinity of the main photolabeled residues.
<p>The structure is shown as a cross section through the luciferin pocket with DSLA bound in the luciferin and ATP sites (2D1S.pdb). Panel A shows a close up view. Panel B is the same view zoomed out (note the 10 Å scale bars) to show the luciferin and ATP pockets. Panel C was obtained by rotating panel B 180° on the y-axis without scaling, viewing the pocket from the opposite side. Cross sections through the protein are capped in white mesh, revealing the residues behind. DSLA is shown with golden carbons. The main photolabeled residues are shown in ball & stick representation with cyan carbons; note that Ser-316 shows two rotamers with each oxygen assigned half occupancy. Important residues for the activity of luciferase that may have been photolabeled are shown with green carbons in stick representation. Hydrogen bonds to ligands and water are shown in panel A by green dashed lines. Surface colors are: white, carbon (except for photolabeled residues, cyan or green); blue, nitrogen; red, oxygen. The upper inset in panel A shows the interaction between Ser-349 and DSLA and the hydrogen bonded water. The lower inset shows the hydrogen bonding network linking the major residues interacting with DSLA through water molecules. Hydrogen bonds shown range from 2.5 to 3.0 Å in length.</p
The 3-Azioctanol inhibits ATP-induced luciferase activity.
<p>Fresh American firefly luciferase pre-incubated with luciferin and increasing concentrations of the alkanol were rapidly mixed with ATP (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#s3" target="_blank">Results</a>). <b>A.</b> Increasing concentrations of 3-azioctanol inhibited the initial phase of light emission. <b>B.</b> The slope of the linear portion of the initial peak of each curve was normalized (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#s3" target="_blank">Results</a>) and plotted against the 3-azioctanol concentrations. Nonlinear least squares fitting yielded an IC<sub>50</sub> value of 220±47 µM and a Hill coefficient of 1.1±0.2.</p
Residues photolabeled in a tryptic fragment of Japanese Firefly Luciferase (340-QGYGLTETT<b>S</b>AIIITPEG<b>D</b>DKPGASGK-366)<sup>a</sup><i><sup>n</sup></i> identified by LTQ–FT mass spectrometry and MSMS.
a–d<p>Same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#pone-0029854-t001" target="_blank">Table 1</a>.</p>e<p>Photoincorporation was either in Ser-249 or Ala-250. The latter residue is rarely labeled by aromatic diazirines.</p
ATP modulates the photoincorporation of [<sup>3</sup>H]-3-Azioctanol into luciferase at equilibrium.
<p>Increasing concentrations of [<sup>3</sup>H]3-azioctanol were preincubated for 30 min with 4.3 µM Japanese firefly luciferase in buffer-A containing 0 (□) or 2 mM (▪) ATP, photolabeled and analyzed as described in the text. Each sample was prepared in triplicate and each point represents the mean and standard deviation. Where error bars are not shown, they are smaller than the symbol. <b>Inset</b>: Luciferase (4.3 µM) pre-incubated with 1 µM [<sup>3</sup>H]3-azioctanol was rapidly mixed with buffer-A containing 0 (white bar) or 2 mM (black bar) ATP, freeze-quenched after 200 ms and photolabeled (see text). Three shots were acquired for each set of condition and the bars represent the mean and the error bars the standard deviation.</p
Identification of photolabeled residues in two peptides.
<p>Conditions are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029854#pone-0029854-g004" target="_blank">Fig. 4</a>. <b>A.</b> TFD–benzyl alcohol (100 µM) photolabels Ser-314 in the peptide Cys-284 – Lys-299. <b>B.</b> 3-azibutanol (1 mM) photolabels Tyr-257 in the peptide Asp-226 – Arg-263.</p
Identification of photolabeled residues in the peptide Tyr-306– Lys-324.
<p>The site of photoincorporation of photolabels was inferred from these MSMS spectrum. In the insets, the predicted charge/mass ratios of ions with an intact N-terminus (b-ions) or C-terminus (y-ions) are shown above and below the sequence, respectively, with the indicated charge. The photolabeled residue is boxed and the experimentally observed values are colored (b-ions in red and y-ions in blue) and in bold and their position indicated on the spectrum. Luciferase was photolabeled after exposure to ATP (2 mM)+photolabel for 200 ms. <b>A.</b> 3-azioctanol (100 µM) photolabels Glu-313. <b>B.</b> TFD-benzyl alcohol (100 µM) photolabels Ser-316. <b>C.</b> TFD-benzyl alcohol (100 µM) photolabels Ile-314.</p
The structure of Japanese luciferase.
<p><b>A.</b> The backbone structure of the highest resolution structure (2D1S.pdb) is shown in ribbon format. The lid domain is shown in gold. The two ligand–binding sites are indicated with arrows and are shown occupied by the concatenated active state analog DSLA. <b>B.</b> The benzothiazole ring of oxyluciferin and DSLA are isosteric. The structures of Japanese luciferase with oxyluciferin +AMP bound (2D1R.pdb) and with the concatenated active state analog DSLA bound (2D1S.pdb) were superimposed. The backbone structures overlay exactly in this region and only that for 2D1R is shown. The ligands' atoms are color coded normally except carbons, which are goldenrod for DSLA and yellow for oxyluciferin and AMP.</p