Weak binding affinity of human 4EHP for mRNA cap analogs

(A) Fluorescence titration curves for binding m⁷GTP to human eIF4E (△), its eIF4EW56Y mutant (●), human 4EHP (■), and fitting residuals. Titrations were carried out in 50 mM HEPES/KOH (pH 7.2), 0.5 mM EDTA, and 1 mM DTT adjusting to I = 150 mM by KCl at 20°C. Protein fluorescence, presented as relative value, was excited at 280 nm and observed at 337 nm. The observed increasing fluorescence signal at a higher concentration of m⁷GTP originates from free-cap analog emission. (B) Graphical comparison of Gibbs free energy of binding (ΔG^o) for association of cap analogs with human eIF4E, its mutant, and human 4EHP, calculated from obtained association constants (K_as).