Fluorescence quenching titration (FQT) binding assay for eIF4E

Murine eukaryotic translation initiation factor 4E (eIF4E, residues 28–217) was expressed and purified as described previously (35). Titration experiments were performed at 20°C in 50 mM Hepes/KOH buffer pH 7.20 containing 100 mM KCl, 0.5 mM EDTA and 1 mM DTT. Aliquots (1 μl) of the tested ligand were added to 1400 μl solution of 0.1 μM eIF4E. Fluorescence of the measured samples was excited at 280 nm (2.5 nm bandwidth) and detected at 337 nm (4 nm bandwidth). Fluorescence intensities were corrected for sample dilution and inner filter effect (36). Equilibrium association constants (K_AS) were determined by fitting the theoretical dependence of fluorescence intensity on the total concentration of the cap analogue to the experimental data points, according to the equation described previously (36). Each experiment was repeated three times and the association constants K_AS were calculated as weighted averages, with the weights taken from reciprocal standard deviations squared. The dissociation constants K_D reported in Table ​Table22 were calculated as K_AS⁻¹.