Fig. 6.
Systematic analyses of FRET between FP–RRs. Phosphorylation was initiated by addition of MgSO4 into the mixture of 20 mM phosphoramidate, 0.6 μM CFP–RR and 2.5 μM YFP–RR. Fluorescence was measured with a plate reader every 450 s for eight times and the FRET ratio changes at the last time point (3150 s) were used to evaluate the FRET for different FP–RR pairs. Tested RRs include all E. coli OmpR/PhoB subfamily RRs and three members of other subfamilies, E. coli NarL, the receiver domain of E. coli NtrC (NtrCn) and Sinorhizobium meliloti FixJ.
A. Time-dependent FRET of RR homo-pairs. Only some of the homo-pairs are shown (refer to Fig. S3 for the rest).
B. FRET between CFP–ArcA and all other YFP–RRs in the presence (grey) or absence (white) of phosphorylation. The data are from four independent experiments and the error bars indicate the standard deviations.
C. FRET between phosphorylated RR pairs. Each coloured square represents one pair of FP–RRs and the diagonal squares from left bottom to right top show the FRET between homo-pairs. The colour of each square indicates the value of FRET ratio change as illustrated by the colour map on the right. The colour map is not continuous linearly in order to display and highlight the pairs with intermediate FRET levels but significantly above the background (0.05–0.2). The data are from four independent experiments while the standard deviations and FRET between non-phosphorylated RRs are shown in Fig. S3. A phylogenetic tree of the E. coli OmpR/PhoB subfamily RRs is shown on the left to indicate the sequence similarity between individual RRs. The phylogenetic tree was generated by clustalx from the alignment of full-length RR sequences (Thompson et al., 1997).
D. Competition of ArcA with the interaction between CFP–ArcA and YFP–CpxR. ArcA protein was titrated into the sample containing phosphorylated CFP–ArcA (0.6 μM) and YFP–CpxR (2.5 μM) followed by the measurement of fluorescence with a fluorometer. The decrease of FRET ratio was calculated by subtracting the ratio of phosphorylated pairs without ArcA present.
E. Interactions of full-length CpxR (CFP–CpxR, white) and the N-terminal receiver domain of CpxR (CFP–CpxRn, grey) with different RRs. Protein concentrations are 0.6 μM CFP–RR and 2.5 μM YFP–RR and fluorescence was measured with the fluorescence plate reader. As the yellow to cyan ratio of CFP–CpxR and CFP–CpxRn decreased upon phosphorylation, all the FRET ratios were corrected for this decrease as shown in Fig. S4.