Figure 6
From: Conformational rearrangements of the C1 ring in KaiC measure the timing of assembly with KaiB
The ATP hydrolysis-coupled rearrangements of the C1 ring occurs before KaiB binding. (a) Time courses of apparent fluorescence intensity (FIapp) of KaiCS157W-D/E in the presence of various concentrations of KaiB. Each plot was vertically shifted for clarity of presentation. (b) and (c) KaiB concentration dependences of total FIapp change (b) and inverse of the half-life time (1/t1/2) (c). The total FIapp change was determined as the difference in FIapp between the first and last time points. Values are means ± S.E. from three or more independent experiments. (d) Time course of FIapp for KaiCS157W-D/E upon addition of KaiB in the presence of 1 mM AMP-PNP (open circle) instead of 1 mM ATP (dashed line). (e) Gel-filtration chromatograms of mixtures containing KaiCS157W-D/E and KaiB in the presence of AMP-PNP. Samples were prepared by mixing KaiB and KaiCS157W-D/E, the latter of which was pre-incubated at 30 °C for 2 h after exchange of ATP with AMP-PNP. The mixtures were loaded onto a column immediately (dashed line) or after 18-h incubation at 30 °C (solid line). A blue line indicates the elution volume of the peak g in Fig. 5b. (f) SDS-PAGE analysis of peaks a’ and b’ in (e) and g in Fig. 5b. Raw image is shown in Supplementary Fig. S6.
