FIGURE 2.

Interaction analysis of MtrA. (A) Analysis of MtrA interaction with AcP. MST measurements for determining interaction affinity of AcP with MtrA (n = 3). (B) SDS-PAGE analysis of purified fluorescently tagged fusion proteins. The top panels represent the fluorescence gel image of various tagged proteins used for FRET studies. Left, GFP and MtrB-GFP; right, MtrA-RFP and RFP. Bottom panels, corresponding CBB stained gels. (C) Effect of acetylation on the interaction between MtrB-GFP and MtrB-RFP (solid lines) or RFP alone (dotted lines) performed at various concentrations of acceptor molecule (1–10 μM) by FRET. RFP protein alone was used as a negative control. FRET analysis was performed by measuring FRET (DA [donor excitation and acceptor emission]/DD [donor excitation donor emission]. (D) Effect of acetylation and phosphorylation on the interaction between MtrB-GFP and MtrA-RFP as measured by FRET. RFP protein alone was used as a negative control. (E) Phosphotransfer time course to analyze the effect of acetylation on MtrA phosphorylation from its cognate SK, MtrB. The assay was performed using (i) wild-type MtrA, (ii) acetylated MtrA (with AcP). (M, marker). 1st panel, autoradiogram; 2nd panel, CBB stained gel; and 3rd panel, the image of blot probed for acetylated lysine. (F) Quantitative measurement of MtrA phosphorylation through sensor kinase, MtrB at various time points (data from experiments reported in E). Signal recorded for unacetylated MtrA at 5-min time point taken as 1, and the rest were normalized to that.