Summary of amide H/D exchange in pepsin digest fragments from SigH (Deuterium exchange time = 10 min)^a.

a<p>Average number of deuterons exchanged determined following 10-min deuterium exchange. Values reported are the mean and standard deviation from at least two independent experiments.</p

HDX-MS of RshA.

<p>(<b>A</b>) Sequence coverage map for RshA. Solid line denotes the peptic fragments analyzed in the study with total sequence coverage of 88%. (<b>B</b>) ESI-Q-TOF mass spectra for one pepsin digest fragment of RshA (35–57) m/z = 881.084, z = 3, which showed significant difference upon RshA binding. (i) Undeuterated RshA peptide (ii) The isotopic envelop for the same peptide from free RshA following 10min deuteration; (iii) The isotopic envelope for the same peptide from RshA and SigH complex following 10 min deuteration., The isotopic envelope for the same peptide. (<b>C</b>) The protein is shown in magenta. The region in red represents regions showing decreased exchange upon interactions with its partner, SigH.</p

HDX-MS of SigH.

<p>(<b>A</b>) Sequence coverage map for SigH. Solid line denotes the pepsin digest fragments analyzed in the study with a total sequence coverage of 71%. (<b>B</b>) ESI-Q-TOF mass spectra for 2 pepsin digest fragments of SigH, (i) (157–171); m/z = 815.89, z = 2 and (ii)* (1–25); m/z = 1259.56, z = 2 which showed significant differences in exchange upon RshA binding. (I) Undeuterated peptide (II) The isotopic envelope for the same peptide from free SigH following 10 min deuteration; (III) The isotopic envelope for the same peptide from SigH and RshA complex following 10 min deuteration. *- The default display of the isotopic envelope for this peptide (1–25); m/z = 1259.56 from the mass spectrometry program, HDX Browser was in color. For clearer display, spectra were highlighted in black (<b>C</b>) The SigH model was prepared using the program I-Tasser using the homologous structure of SigR from <i>Streptomyces coelicolor</i>. The protein is shown in green. The regions in red represent regions of the SigH protein showing decreased deuterium exchange in the presence of its interacting partner, RshA.</p

Estimation of iron in RshA.

<p>Estimation of iron in RshA.</p

Model of interaction.

<p>A model for the interaction between SigH and RshA is proposed through a docking model, which was prepared with the help of the program Z-Dock <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043676#pone.0043676-Chen1" target="_blank">[52]</a>. The proteins follow the respective coloring schemes, as in Figs. 2d and 3d.</p

Summary of amide H/D exchange in pepsin digest fragments from RshA (Deuterium exchange time = 10 min)^a.

a<p>Average number of deuterons exchanged determined following 10-min deuterium exchange. Values reported are the mean and standard deviation from at least two independent experiments.</p

Targeted mutagenesis based on HDX-MS.

<p>Soluble fractions of two cell lysates (5.0 mg crude protein each RshA and mutants/SigH and mutants) were mixed together in a buffer containing 50 mM Tris HCl (pH 7.5), 100 mM NaCl, 10 mM DTT and 0.01% X-100 and allowed to interact at 4°C for 3 h on a rocker platform. DTT was periodically replenished during interaction. After interaction the protein mix was mixed with pre-swollen glutathione beads for 2 h at 22°C and then the beads were washed once each with 50 mM Tris HCl (pH 7.5), 10 mM DTT, 0.01% Triton X-100, 100 mM NaCl; 10 mM DTT, 0.01% Triton X-100, 50 mM Tris HCl (pH 7.5), 200 mM NaCl; 50 mM Tris HCl (pH 7.5), 300 mM NaCl; 10 mM DTT, 0.01% Triton X-100. The washed resin was mixed with SDS gel-loading buffer and the bound proteins were resolved on 15% SDS-PAGE and stained with Coomassie blue or processed for immunoblotting. (<b>A</b>) SDS-PAGE gel of a GST-pull down assay showing the interaction of WT RshA and WT SigH and their mutants. The gel was stained with Coomassie blue and another identical gel was used for Western Blot using anti 6xHis antibody. (<b>B</b>) Western blot using a 6xHis antibody in a GST-pull down assay to study the interaction of SigH and RshA. Both panel b and C have identical loading, however panel B represents SigH and and its mutants while panel C represents RshA and its mutants. (<b>i</b>) WT or mutant GST:RshA and 6xHis:SigH proteins were used. (<b>ii</b>) In the lower panel, WT or mutant GST:SigH and 6xHis:RshA proteins were used. (<b>C</b>) RshA-SigH interaction does not require metal ion. Purified 5 µM RshA/SigH each was mixed in the presence of 50 mM Tris HCl (pH 8.0), 100 mM NaCl, 0.01% Triton X-100 at 25°C. Same samples were loaded in both the native gel (ph 8.8) and SDS-PAGE and stained with Coomassie blue. The left panel is native gel and the right panel is SDS-PAGE. (<b>D</b>) Intrinsic tryptophan fluorescence of RshA (oxidized), SigH and their complex. 3 µM of each protein was mixed in a buffer containing 50 mM Tris HCl (pH 8.0), 100 mM NaCl, 0.01% Triton X-100 at 25°C for 2 hr to make the SigH-RshA complex. RshA has a single tryptophan while SigH has two tryptophan residues. The differences in the spectra clearly show that the complex formation is a dynamic process.</p