LC-MS analysis of (apo)flavodoxin.

<p>Monomeric protein species are labelled M. Molecular masses of NBD-Cl and NBD are 199.6 and 164.1 Da, respectively. Expected mass and measured mass are average masses.</p

Cartoon drawing of the X-ray structure of flavodoxin from <i>A. vinelandii</i>.

<p>α-Helices are shown in red, β-strands in blue and loops in white. The FMN cofactor is coloured yellow and the backbone of residue 69 is coloured magenta. Note that this residue is in immediate vicinity of FMN. The X-ray structure is of the C69A variant of the protein (pdb ID 1YOB) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041363#pone.0041363-Alagaratnam1" target="_blank">[30]</a>, in which the single cysteine at position 69 is replaced by alanine. This protein variant is largely similar to flavodoxin regarding both redox potential of holoprotein and stability of apoprotein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041363#pone.0041363-Steensma1" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041363#pone.0041363-vanMierlo2" target="_blank">[54]</a>.</p

Reactions of NBD-Cl with protein cysteine and sulfenic acid states.

<p>Schematic diagram showing steps involved in reaction of NBD-Cl with cysteine and sulfenic acid.</p

Native mass spectrometry of (apo)flavodoxin.

<p>Monomeric protein species are labelled M, Molecular masses of FMN is 455.3 Da. Expected mass and measured mass are average masses.</p

Superdex 75 size exclusion chromatography elution profiles of apoflavodoxin.

<p>(A) Apoflavodoxin (38 μM). (B) Apoflavodoxin (38 μM) after 22 h incubation with 10 mM H₂O₂ at 4°C. (C) C69A apoflavodoxin (79 μM). (D) C69A apoflavodoxin (79 μM) after 24 h incubation with 10 mM H₂O₂ at 4°C. Flow rate is 0.5 mL/min and temperature is 25°C.</p

MonoQ anion exchange chromatography elution profiles of flavodoxin and apoflavodoxin.

<p>(A) Flavodoxin (40 μM). (B) Flavodoxin (40 μM), kept in the presence of 10 mM DTT for a period of 10 min. (C) Apoflavodoxin (50 μM). (D) Apoflavodoxin (50 μM), kept in the presence of 10 mM DTT for a period of 10 min. Gradient composition: buffer A is 25 mM Tris-HCl pH 8.0, and buffer B is 25 mM Tris-HCl pH 8.0, containing 1 M KCl. Flow rate is 1.0 mL/min. Dashed lines show conductivities of elution buffers. The molecule eluting at 7 mL is DTT. Temperature is 25°C.</p

Oxidation states of protein cysteines, and their reversibility by DTT.

<p>Schematic diagram showing steps involved in hydrogen peroxide-induced oxidation and DTT-induced reduction of protein cysteines.</p

Monitoring of (apo)flavodoxin under H₂O₂-induced oxidative stress by LC-MS.

<p>For clarity, the zoomed in LC-MS regions that display the +10 charge state of monomer and the +19 charge state of dimer are shown (i.e., m/z range of 1940 to 2070); however, analysis is done on the whole m/z range. (A) Apoflavodoxin. (B) Apoflavodoxin incubated for 30 min with 10 mM H₂O₂. (C) Apoflavodoxin incubated for 30 min with 100 mM H₂O₂. (D) Apoflavodoxin incubated for 30 min with 200 µM NBD-Cl and 100 mM H₂O₂. (E) Flavodoxin incubated for 30 min with 190 µM NBD-Cl and 100 mM H₂O₂. Protein concentration is 5 µM and incubations were done at room temperature. M represents apoflavodoxin monomer with non-oxidised thiol; MO, MO2 and MO3 are the sulfenic, sulfinic and sulfonic acid states of apoflavodoxin, respectively. M-NBD is monomer protein with the thiol adduct of NBD-Cl, MO-NBD is monomer protein with the sulfenic acid adduct of NBD-Cl, and D represents disulfide-linked apoflavodoxin dimer.</p

Spectroscopic characteristics of apoflavodoxin modified with NBD.

<p>Apoprotein (20 μM) before (solid line) and after (dashed line) incubation with 200 μM NBD-Cl for 1 hour. Modified protein absorbs maximally at 420 nm, which is characteristic for the presence of a thiol-NBD conjugate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041363#pone.0041363-Ellis1" target="_blank">[47]</a>.</p

Kid–Kis complexes (molar ratio of 1:1) interact tightly with region I

<p><b>Copyright information:</b></p><p>Taken from "Interactions of Kid–Kis toxin–antitoxin complexes with the operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid–Kis oligomers"</p><p></p><p>Nucleic Acids Research 2007;35(5):1737-1749.</p><p>Published online 21 Feb 2007</p><p>PMCID:PMC1865072.</p><p>© 2007 The Author(s)</p> Macromolecular native mass spectrometry was performed on Kid–Kis and on Kid–Kis– DNA complexes in ammonium acetate (50 mM), pH 5.8. () Mass spectrum of a mixture of Kid:Kis at a molar ratio of 1:1 (Kis 15 μM) and () and () mass spectra of Kid:Kis: DNA mixtures at molar ratios of 40:40:1 and 10:10:1 (Kis 15 μM), respectively. Kid and Kis are indicated with blue rectangles and orange ellipses, respectively, and the DNA fragment with double strand. Each complex is represented by an appropriate combination of rectangles, ellipses and/or DNA double strand. Molecular masses and relative amounts of complexes are shown in Supplementary Tables 1 and 2, respectively