Stopped-flow and Brownian dynamics studies of electrostatic effects in the kinetics 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 degrees C, in 50 mM Hepes-KOH buffer, pH 7.2, and 50, 150 and 350 mM KCl, was measured using a stopped-flow spectrofluorometer, and was simulated by means of a Brownian dynamics method. For most of the stopped-flow measurements a single bimolecular step is an inadequate description of the binding mechanism and an additional step is required to accommodate the kinetic data. The rate constants derived from assumed one-step and two-step binding models were determined. The forward rate constants towards the complex formation decrease, and the reverse rate constants increase, with increasing ionic strength. The association rate constants derived from the stopped-flow measurements and the computed diffusional encounter rate constants agree, indicating that the first observed step can be viewed as a diffusionally controlled encounter of the protein and the ligand. Moreover, comparison of experimental and computed bimolecular association rate constants indicate that the experimentally observed decrease of the rate constants with the increasing ionic strength is caused by two factors. 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 strengths the ligand must be closer to the binding site to induce the fluorescence quenching.
Błachut-Okrasińska
E
E
Department of Biophysics, University of Warsaw, Poland.
Bojarska
E
E
Niedźwiecka
A
A
Chlebicka
L
L
Darzynkiewicz
E
E
Stolarski
R
R
St piński
J
J
Antosiewicz
J M
JM
D000818:Animals; D015226:Dinucleoside Phosphates / Q000737:chemistry* / Q000378:metabolism*; D039561:Eukaryotic Initiation Factor-4E; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012315:RNA Caps; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D055672:Static Electricity
D015226:Dinucleoside Phosphates; D039561:Eukaryotic Initiation Factor-4E; D008024:Ligands; D010448:Peptide Initiation Factors; D012315:RNA Caps; D011994:Recombinant Proteins; C062229:7-methyl-diguanosine triphosphate