Fig. 2: Quantitative analyses of the BphP/RR interactions.
a Size-exclusion chromatography (SEC) of the EGFP-labeled response regulators DrRR and AtRR1 in the absence (top) and presence (bottom) of DrBphP and Agp1, respectively. Vertical dashed lines indicate the retention volume of free RR monomer. Top panels: In isolation, EGFP-DrRR (45.4 kDa) and EGFP-AtRR1 (45.4 kDa) eluted as a monomer and a monomer/dimer mixture, respectively. DrBphP (84.0 kDa) and Agp1 (83.8 kDa) are known to be dimers, and their apparently high molecular weights (~300 kDa) can be explained by the poor resolution of large proteins in the conditions. Bottom panels: When combined with DrBphP in its dark-adapted state (D) or after 655-nm illumination (R), the profile for EGFP-DrRR shifted to shorter retention times, indicative of interactions. By contrast, addition of Agp1 had little effect on the retention of AtRR1. The top panels are plotted at 280 nm and the bottom panels at 489 nm. The A489 signals from BphPs were negligible. b Isothermal titration calorimetry (ITC) measurements. Differential power (DP) resulting from injections of the response regulator to the BphP is plotted against time, and the binding enthalpy (ΔH) is plotted against the molar ratio of the proteins. See Supplementary Fig. 2 for additional data and control measurements. c Surface plasmon resonance (SPR) measurements. The response regulators were coupled on the sensor surface, and varying concentrations of the corresponding phytochrome were applied in darkness (D) or after red-light illumination (R). The sensorgrams (black lines), the kinetic fits (blue lines), and the parts of the data that were used for kinetic analysis (orange) are indicated. For kinetic analyses, DrBphP concentrations of 8–67 µM were used. Green lines mark the Req values that were used for evaluating the steady-state affinity data, shown in Supplementary Fig. 2i. Inset: Steady-state fit of the concentration series, where Req values were used for affinity approximation. See Supplementary Fig. 2i for a table of SPR fitting values. Source data are provided as a Source data file.
