Figure. 5.

Mutant sensor construction and characterization. (a) Bar graph showing the species-weighted average fluorescence-lifetime of the CB wild-type, single-mutant (sm), and double-mutant (dm) FRET sensors before (F1_D0, F1_smD0, and F1_dmD0) and after FlAsH labeling (F1_DA, F1_smDA, and F1_dmDA), and the FlAsH-labeled sensors in the presence of a 100-fold molar excess of GephFL. Data from three different batches of experiments are presented as mean values ± SD. (b) GephFL binding affinity plot of F1_smDA (orange) and F1_dmDA (wine). Binding affinities were determined by first converting ⟨τ⟩_x into the fractional saturation using Eq. 4 and the data were further fitted with Eq. 5. The GephFL binding affinity constant (K_D) for F1_smDA and F1_dmDA were measured as 47 ± 14 and 43 ± 11 μM, respectively. Data from three different batches of experiments are presented as mean values ± SD. (c) Model-free description of the interfluorophore distance distribution underlying the time-resolved fluorescence intensities (Eqs. 12 and 13). Normalized distance distribution curves shown for F1_smDA and F1_dmDA in the absence and presence of GephFL. F1_smDA and F1_dmDA show a major peak at 28 Å and a shoulder at 36 Å, which is significantly different from F1_DA, particularly for the major peak located in this case at ∼43 Å (Fig. S9d). Upon complexation with GephFL the major peak shifts to 36 Å with a weak shoulder at ∼28 Å.