Figure 8.

Kinetics of CheA autophosphorylation with Tar variants. E. coli CheA, CheW, and Tar_FO 4Q and short or Tar_SC (in a 1:1:3 subunit ratio, 1 μM CheA note that the receptor subunit is a single-chain “dimer”) were allowed to complex at 25 °C for 1 h prior to exposure to [γ-³²P]ATP for the indicated time points. Each data point represents an average over two to four assays. (A) CheA-P formation over time in the presence of CheW and Tar variants. The inset shows CheA-P buildup with Tar_FO short compared to CheA:CheW alone out to 30 min. Curves were fit to a first-order kinetic transition (see Experimental Procedures). (B) Addition of cold ADP to CheA and CheW with or without Tar variants after initial autophosphorylation with [γ-³²P]ATP for 6 min. (C) Addition of cold ATP to CheA and CheW after incubation with [γ-³²P]ATP for 6 min. (D) Transfer to CheY in the presence of CheA and CheW with or without Tar_FO 4Q and short. Error bars represent the standard error of the mean (SEM) calculated from three independent experiments (n = 3).