Fig. 1: Purification and biochemical characterization of DosP.

a Domain organization of DosP over its linear amino acid sequence. b Schematic for the MBP-DosP expression construct. TEV protease recognition sequence is shown under brackets. Cleavage site marked by arrow and resulting N-terminal methionine corresponds to DosP M1. c Coomassie-stained 4-15% SDS-PAGE analysis of purified DosP from the indicated steps. Migration positions of the proteins are indicated with arrows. Molecular weight standards are labeled. d SEC purification of free DosP, MBP, and TEV protease following proteolysis of MBP-DosP using a 10/300 Superose 6 gel filtration column as described in Methods. e Absorption spectra of DosP^WT under aerobic conditions (red), compared to anaerobic conditions (blue). f Absorption spectra of DosP^R97A under aerobic conditions (red), compared to anaerobic DosP^R97A (blue). g Turnover of 2 µM c-di-GMP at 25 °C by 10 nM protein: aerobic DosP^WT (open red triangles), anaerobic DosP^WT (open blue squares), aerobic DosP^R97A (filled red triangles), and anaerobic DosP^R97A (filled blue squares). Black line indicates time regime used to determine k_cat. Each data point represents a measurement from an independent experiment (n = 2). The assay buffer was 20 mM Tris-HCl, 2.5 mM MgCl₂, 2.0 mM DTT, pH 8.0. h kcat values determined from g. Values are the averages of K_cat measurements from two independent experiments. Source data are provided as a Source Data file.