Ion Exchange in the Nonspecific Bimolecular Association and the Unimolecular DNA Bending in Specific Binding of IHF to DNA

Abstract

Protein-DNA interactions are strongly modulated by salt. Previous studies on the equilibrium binding of a cognate DNA sequence H′ to integration host factor (IHF), an architectural protein from E. coli that bends its cognate site by nearly 180°, have shown that the slope SKD of ∼8 on a log(KD) versus log([KCl]) plot depends on the anion type, suggesting that both release of counterions from the DNA and the uptake and release of ions from the protein must be playing a role. Here, we probe the effect of [KCl] on the bimolecular association/dissociation as well as the unimolecular bending/unbending rates, by monitoring the relaxation kinetics of the complex between IHF and ∼35-bp long H' substrate end-labeled with a FRET pair, in response to a laser temperature-jump. Our results and analysis reveal two notable results. First, that the unimolecular bending step is nearly independent of [KCl]. Second, that the bulk of the salt-dependence appears in the nonspecific association/dissociation step, with the equilibrium constant for that step accounting for more than half of the total SKD observed. The latter result is in contrast to what one might expect if counterion release from the DNA was the dominant contribution to the salt-dependence, since the extent to which the H' substrate makes contact with the protein in the fully wrapped specific complex is significantly greater than in the nonspecific complex. One possible scenario is that counterion release is the dominant term in the formation of the nonspecific complex, whereas in the transition from the nonspecific to the specific complex, the extent of the counterion release is masked by the uptake and release of ions by the protein, as a result of conformational rearrangements in the protein.