Fig. 5. Kinetics and thermodynamics of nucleotide binding to Rel_Bs in the presence and absence of DarB.

(A) Kinetics of MANT-GDP binding to Rel_Bs^NTD (in red) and DarB:Rel_Bs^NTD (in blue) monitored by stopped flow. The interaction was measured by FRET excitation of MANT fluorescence upon mixing 10 μM protein with increasing concentrations of MANT-GDP (B). (C) Kinetics of MANT-GDP dissociation from Rel_Bs^NTD (in red) and DarB:Rel_Bs^NTD (in blue). (D) Kinetics of MANT-ATP binding to Rel_Bs^NTD (in red) and DarB:Rel_Bs^NTD (in blue). The interaction was measured as in (A) by FRET excitation of MANT-ATP fluorescence upon mixing 10 μM protein with increasing concentrations of MANT-ATP (E). (F) Kinetics of MANT-ATP dissociation from Rel_Bs^NTD (in red) and DarB:Rel_Bs^NTD (in blue). In both cases, the dissociation was monitored upon rapid mixing with an excess (2 mM) of unlabeled GDP or ATP. Binding of APCPP to Rel_Bs^NTD (G) and DarB:Rel_Bs^NTD (H) monitored by ITC.