Figure 5: Comparison of labeled, non-phosphorylated and phosphorylated states of FGFR1 KD by smFRET. | Scientific Reports

Figure 5: Comparison of labeled, non-phosphorylated and phosphorylated states of FGFR1 KD by smFRET.

From: Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

Figure 5

(a,b) EPR-S 2D histogram plots with projections (bottom: EPR; right: S) of double-labeled H589BCNK.TC (a) and L662BCNK.TC (b) in non-phosphorylated (N) and phosphorylated (P) states for comparison. The 1D-histogram subplots (grey bars) are compared to the Gaussian distribution fits (red lines, subpopulations in blue). The EPR 1D histogram subplots show a unimodal distribution of events (counts) in all measured samples except for L662BCNK.TC in the non-phosphorylated form, which is described by a Gaussian Mixture with two components (0.8 and 0.2 respectively) shown in blue. (c) EPR mean from 3 independent experiments under phosphorylating (P, orange), non-phosphorylating (N, blue) or control, ADP-supplemented (C, purple) conditions. Error bars indicate the standard deviations (SDs). Statistical significance was assessed using one-way ANOVA with Bonferroni multiple comparison test (**0.001 < P < 0.01, ***0.0001 < P < 0.001). (d) TR-FRET readout of FlAsH-EDT2 and TAMRA-X labeled H589BCNK.TC (left) and L662BCNK.TC (right), comparing non-phosphorylated (N) and phosphorylated (P) samples. The fluorescent lifetime decay of the FRET donor is determined by fitting into a stretched exponential decay model with the measured instrument response function (IRF). See also Supplementary Fig. S5.

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