Stopped-flow and Brownian dynamics studies of electrostatic effects in thekinetics of binding of 7-methyl-GpppG to the protein eIF4E

The kinetics of binding 7-methyl-GpppG, an analogue of the 5'-mRNA cap, to the cap-binding protein eIF4E, at 20 degreesC, in 50 mM Hepes-KOH buffer, p H 7.2, and 50, 150 and 350 mM KCl, was measured using a stopped-flow spectr ofluorometer, and was simulated by means of a Brownian dynamics method. For most of the stopped-flow measurements a single bimolecular step is an inad equate description of the binding mechanism and an additional step is requi red to accommodate the kinetic data. The rate constants derived from assume d one-step and two-step binding models were determined. The forward rate co nstants towards the complex formation decrease, and the reverse rate consta nts increase, with increasing ionic strength. The association rate constant s derived from the stopped-flow measurements and the computed diffusional e ncounter rate constants agree, indicating that the first observed step can be viewed as a diffusionally controlled encounter of the protein and the li gand. Moreover, comparison of experimental and computed bimolecular associa tion rate constants indicate that the experimentally observed decrease of t he rate constants with the increasing ionic strength is caused by two facto rs. The first is less effective steering of the ligand towards the binding site at higher ionic strengths, and the second is that for higher ionic str engths the ligand must be closer to the binding site to induce the fluoresc ence quenching.