Fig. 2

In-source collision-induced dissociation of WhiB1 and isolation of a holo-WhiB1:σ^A complex. a Deconvoluted ESI-MS spectra of [4Fe-4S] WhiB1 (black spectrum). The [4Fe-4S] cluster bound form is the only significant species. Application of isCID with increasing energy (up to 140 eV, red spectrum, intermediate energies gave spectra in gray) resulted in detection of cluster fragment species, along with apo-WhiB1 and sulfur adducts, as indicated. Asterisks indicate oxygen adducts and triangles indicate sodium adducts (in addition to those indicated as major species). Note that sodium adducts increase with isCID. Peaks annotated with mass numbers could not be unambiguously assigned. WhiB1 (20 μM [4Fe-4S]) was in 250 mM ammonium acetate, pH 8.0. A reaction scheme based on the mass spectrometry data is shown below the spectra. b Absorbance spectrum of the protein complex isolated from cell extracts of E. coli transformed with plasmids expressing His-tagged M. tuberculosis σ^A (His-σ^A) and untagged WhiB1 by nickel affinity chromatography. c Gel filtration elution profiles (280 nm) of His-σ^A (red trace), WhiB1 (purple trace), His-σ^A-WhiB1 (blue trace), and His-σ^A-WhiB1 after exposure to NO (black trace). d SDS-PAGE analysis of the fractionated NO-treated His-σ^A-WhiB1 (black trace in c) as follows: lane 1, protein molecular weight markers; lane 2, nitrosylated His-σ^A-WhiB1 sample applied to the column; lanes 3–5, species eluting ~42 ml; lanes 6 and 7, species eluting at ~56 ml; lanes 8–10, species eluting at ~93 ml. e UV-visible spectrum of fractions eluting at ~42 ml (upper panel) and fractions eluting at ~93 ml (lower panel). f SDS-PAGE analysis of WhiB1:His-σ^A complexes. Lane 1, molecular weight markers; lane 2, isolated WhiB1:His-σ^A; lane 3, cell-free extract of E. coli expressing His-σ^A and WhiB1(Cys40Ala); lane 4, flow through from nickel affinity column after application of the cell-free extract shown in lane 3; lane 5, eluate from the nickel affinity column loaded with the cell-free extract shown in lane 3. The sizes of the standard proteins and the locations of WhiB1 proteins and His-σ^A (σ^A) are indicated