Figure 3.

Full-length gephyrin-mediated CB activation. (a) Average fluorescence lifetime of CFP in F1_D0 (teal), F1_DA alone (cyan), and in the presence of GephFL (black), SH3 domain (orange), and NL2_icd (dark red). ∗∗∗p < 0.005. Data from three different batches of experiments are presented as mean values ± SD. (b) Fluorescence lifetime of CFP in F1_DA and F1_DA-GephFL complexes with increasing concentrations of full-length gephyrin (GephFL, black). Data were scaled to a maximum of 100 for easier comparison. The decay histograms are fitted in two ways: 1) with a multiexponential fitting model (Eq. 1) to determine K_D and 2) with two FRET species (R(i)) with a Gaussian distance distribution (half-widths 5 Å) and a single NoFRET species (see Methods). (c) GephFL binding affinity was determined based on the average fluorescence lifetime converted into the fractional saturation using Eq. 4. The data were fitted with Eq. 5. For the F1_DA-GephFL complex (black) a dissociation constant (K_D) of 4.5 ± 1.7 μM and for the F1_DA-SH3 complex (orange) a K_D of 373 ± 116 μM (data ± SD) were obtained. Data from three different batches of experiments are presented as mean values ± SD. (d) Plot of the contribution of the two FRET species (high- and low-FRET states) against the concentration of GephFL obtained when analyzing the time-resolved fluorescence intensities with the Gaussian distribution model (Eq. 9). Curves showing the fraction of F1_DA molecules in the closed/high-FRET state (filled squares, black) with a FRET pair distance (R₁) of 25 ± 1.1 Å and their gradual transition into the open/low-FRET state (open squares) exhibiting a FRET pair distance (R₂) of 46 ± 1.5 Å upon addition of GephFL. The transition of F1_DA from the high- to the low-FRET state is illustrated in cartoon representation.