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. 2013 Sep 6;288(42):30473–30484. doi: 10.1074/jbc.M113.489914

FIGURE 2.

FIGURE 2.

Functional analysis of AtsR domains. A, schematic domain organization of AtsR and its derivatives (domains are not drawn to scale). The predicted sites of phosphorylation are His-245 (H245) and Asp-536 (D536). TM, transmembrane domain; HK, histidine kinase domain; ATPase, ATPase domain; RD, receiver domain; A, alanine. B, In vitro phosphorylation assays. Five μmol of purified AtsR, AtsRH245A, AtsR-HK, AtsR-RD, AtsR-HK and AtsR-RD, AtsRH245A and AtsR-RD, and AtsR-HK and AtsR-RDD536A were added in a standard phosphorylation mixture (100 mm Tris-HCl, pH 8, 50 mm KCl, 5 mm MgCl2, 1 mm DTT, 5 μCi [γ-33P]ATP) and incubated for 15 min at 25 °C. Reactions were terminated by adding 3× SDS-PAGE loading buffer and resolved by SDS-PAGE. The phosphorylated proteins were visualized using a PhosphorImager (top). Phosphorylated and non-phosphorylated proteins were detected by Coomassie Blue staining (bottom). The location of phosphorylated bands of the AtsR, AtsR-HK, and AtsR-RD proteins are denoted with arrows. C, chemical stability of phosphorylated proteins. Phosphorylated AtsR (lanes 1, 3, and 5) and AtsRD536A (lanes 2, 4, and 6) were treated with 1 m NaOH or 1 m HCl or were left untreated for 45 min at room temperature. The reactions were neutralized with 0.25 volumes of 2 m Tris, pH 8, and analyzed by PhosphorImager after SDS-PAGE.