Fig. 5. Efficacies of FS and FS analogs at stimulating direct MANT-GTP fluorescence and at inducing FRET in C1/C2.

Fluorescence experiments were performed as illustrated in Fig. 4. Data were normalized relative to the effect of FS (100 μM, = 100%). All FS analogs were used at a concentration of 100 μM. Panel A shows normalized direct fluorescence increase of MANT-GTP at λ_ex = 350 nm and λ_em = 420 nm induced by FS and FS derivatives in the presence of 10 mM Mn²⁺. Direct fluorescence was calculated subtracting basal fluorescence at λ_em = 420 nm (C1/C2 + MANT-GTP) from the corresponding fluorescence determined in the presence of FS analogs. Panel B shows normalized FRET from Trp1020 to MANT-GTP induced by FS and FS derivatives at λ_ex = 280 nm and λ_em = 420 nm in the presence of 10 mM Mn²⁺. FRET was calculated by subtraction of the F420/F335 ratios of basal fluorescence from the corresponding ratio in the presence of FS analogs. Data from three to five experiments with at least two independent batches of protein were pooled and are shown as means ± SD. Statistical comparisons between FS and different analogs were performed using the t-test. * p < 0.05 and ** p < 0.01.