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. 2022 Sep 16;2(4):100079. doi: 10.1016/j.bpr.2022.100079

Figure 4.

Figure 4

Gephyrin E domain mediates CB activation. (a) Bar graph showing the observed species-weighted average fluorescence lifetime of CFP in F1DA (cyan) and in the presence of GephG (purple), GephE (olive), GephLE (magenta), GephFL (black), and the monomeric E domain variant (GephEmm, blue). ∗∗p < 0.05, ∗∗∗p < 0.005; ns, statistically not significant. (b) Binding affinity curves for GephLE (magenta), GephE (olive), and GephEmm (blue) determined based on the average fluorescence lifetime converted into fractional saturation using Eq. 5. Curves are fitted with Eq. 8 (see Methods) to determine the KD for GephLE, GephE, and GephEmm. (c) Plot of the contribution of the two FRET species (high-FRET state and low-FRET state) against the concentrations of GephLE (triangle) and GephE (circle) obtained when analyzing the time-resolved fluorescence intensities with the Gaussian distribution model (Eq. 9). The high-FRET state (R1, filled circle and triangle) decreases, and the low-FRET (R2, open circle and triangle) state increases with increasing concentrations of both ligands. (a - c) Data from three different batches of experiments are presented as mean values ± SD.