Fig. 6. Structures of signal agonists and antagonists analyzed.

Fig. 7. Microcalorimetric titration of NTodS and CTodS with different hydrocarbons. (Upper) Titration of 21 mM CTodS with o-xylene and 20 mM NTodS with 1,2,3-TMB, o-xylene, and o-chlorotoluene. The ligand concentration was 1 mM in all cases. For clarity, raw titration data have been shifted arbitrarily on the y axis. (Lower) Integrated and corrected peak areas for the titration of NTodS with 1,2,3-TMB, o-xylene, and o-chlorotoluene. Derived thermodynamic data are given in SI Table 3.

Fig. 8. Autophosphorylation of TodS in the absence and presence of different toluene concentrations. (A) TodS was incubated for 20 min with [³²P]ATP and toluene at the concentrations indicated before separation on SDS/PAGE gels. The first lane corresponds to the basal phosphorylation rate of TodS (absence of toluene). (B) Densitometric analysis of data shown in A. The value corresponding to the basal autophosphorylation (first lane in A) was subtracted from each of the remaining values. Data were then fitted with the Michaelis-Menten equation, and a toluene concentration at half the maximal autophosphorylation activity of 10 ± 2 mM was determined.

Fig. 9. Autophosphorylation of TodS protein and its F79A mutant. Experiments carried out in 50 mM Tris·HCl (pH 7.5)/200 mM KCl/2 mM MgCl₂/0.1 mM EDTA/2 mM DTT; 300 pmol TodS was incubated with 0.2 mM [g-³²P]ATP containing 4 mCi g-³²P in the absence and presence of 100 m M toluene. At the indicated time points, 7.5-ml samples were obtained and analyzed by SDS/PAGE.