Fig. 4.
Intact protein LC–MS analyses of PorXFJ point mutants in the absence or presence of phosphorylation in vitro. The blue and red spectra correspond to nonphosphorylated and phosphorylated versions of the protein, respectively. Comparing the theoretical and observed masses (inset blue and black numbers, respectively, in dalton) showed that the acetyl phosphate (AcP) phosphodonor promotes the phosphorylation of Asp54 and/or Thr271 (“+1 Pi” or “+2 Pi” labels). Alternatively, AcP was found to induce cyclization of Asp54 and Lys104, resulting in a dehydration reaction (“− 1 H20” labels) and subsequent phosphorylation at Thr271 only (“− 1 H20, + 1 Pi” labels).