A slow conformational change of BtuC2D2 measured by quenching of tryptophan fluorescence. (A) BtuC2D2 was incubated for 0 (black), 1 (red), 3 (magenta) or 7 hours (blue) under the working conditions. ATP was then removed by desalting and all samples were diluted to the same concentration. 50 μL of a 1 μM protein solution were excited at 230–300 nm, emission was measured at 330 nm. (B) BtuC2D2 was incubated for 16 hours under the working or the ATP-bound conditions, as indicated. Fresh protein is shown for comparison as well. ATP was removed by desalting and 50 μL of 1 μM were excited at 275 nm (peak absorbance for BtuC2D2), emission was measured at 330 nm. The experiments were repeated at least three times and error bars represent standard deviations of technical triplicates of a single experiment. Also shown are P-values obtained by a Student’s t-test (ns, not significant).