Quantitative monitoring of His and Asp phosphorylation in a bacterial signaling system by using Phos‐tag Magenta/Cyan fluorescent dyes

• Published in: Electrophoresis 2019-11, Vol.40 (22), p.3005-3013
• Main Authors: Kinoshita‐Kikuta, Emiko, Kusamoto, Hiroshi, Ono, Syogo, Akayama, Keisuke, Eguchi, Yoko, Igarashi, Masayuki, Okajima, Toshihide, Utsumi, Ryutaro, Kinoshita, Eiji, Koike, Tohru
• Format: Article
• Language: English
• Subjects: Adenosine triphosphate; Aspartic Acid - analysis; Aspartic Acid - metabolism; Bacterial Outer Membrane Proteins - metabolism; Bacterial Proteins - metabolism; Dyes; E coli; Escherichia coli Proteins - metabolism; Fluorescent dyes; Fluorescent Dyes - analysis; Fluorescent Dyes - metabolism; Histidine; Histidine - analysis; Histidine - metabolism; Histidine kinase inhibitor; Kinases; Monitoring; Multienzyme Complexes - metabolism; Phosphoproteins - metabolism; Phosphorylation; Phosphorylation - physiology; Phosphorylation assay; Phos‐tag dye technology; Protein phosphorylation; Quantitative analysis; Signal Transduction - physiology; Signalling systems; Staining; Trans-Activators - metabolism; Two‐component system
• ISSN: 0173-0835; 1522-2683
• DOI: 10.1002/elps.201900261

In the bacterial signaling mechanisms known as two‐component systems (TCSs), signals are generally conveyed by means of a His–Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation status. Here, we show that the Phos‐tag dye technology is suitable for the fluorescent detection of His‐ and Asp‐phosphorylated proteins separated by SDS‐PAGE. The dynamics of the His–Asp phosphorelay of recombinant EnvZ‐OmpR, a TCS derived from Escherichia coli, were examined by SDS‐PAGE followed by simple rapid staining with Phos‐tag Magenta fluorescent dye. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of EnvZ and OmpR in the presence of adenosine triphosphate (ATP) or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from EnvZ to OmpR, which occurs within 1 min in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos‐tag Cyan gel staining. We believe that the Phos‐tag dye technology provides a simple and convenient fluorometric approach for screening of HK inhibitors that have potential as new antimicrobial agents.