(a) Scheme of the IF-to-OF transition by comparing the IFwide and OFopen conformation of TmrAB. TmrA, TmrB, and ATP are shown in cyan, yellow, and red, respectively. (b) Loss in peptide binding after the ATP-induced IF-to-OF transition. TmrAEQB (0.2 µM) in Nds was incubated for 5 min at 45 °C with the R9LQK peptide (10 µM, supplemented with 3H-R9L) in the presence and absence of Mg2+-ATP (1 mM). Peptide binding was monitored by SPA. Signals were background-corrected and multiplied by the dilution factor of radiolabeled peptides. (c) Peptide binding after the IF-to-OF switch. The fluorescence anisotropy of C4F peptide (50 nM) was determined at λex/em = 485/520 nm in the absence and presence of TmrAEQB in Nds (1 µM). To distinguish between the IF and OF conformation, TmrAEQB was incubated for 5 min at 45 °C with and without Mg2+-ATP (1 mM). (d) Nucleotide-dependent peptide binding. TmrAEQB in Nds (1 µM) was incubated for 5 min at 45 °C with increasing concentrations of nucleotide. (e) Equilibrium binding affinity of ATP or ADP analyzed by scintillation proximity assay. TmrAEQB (0.2 µM) was incubated with increasing concentration of ATP or ADP supplemented with 3H-ATP or 3H-ADP, respectively. The mean of three independent experiments was analyzed by a one-site binding model, KD, ATP = 0.10 ± 0.03 mM, KD, ADP = 0.25 ± 0.10 mM. In (b–e) the mean ± SD (n = 3) is displayed.