Thermodynamics of Interactions between Histone-like Proteins from Escherichia coli (HU and IHF) and Intact Duplex DNA

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The structural homologs HUαβ and IHF are major nucleoid associated proteins (NAPs) of Escherichia coli, which organize chromosomal DNA and facilitate numerous DNA transactions. Using isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) and a series of DNA duplex lengths (8, 15, 34, 38 and 160 base pairs) we establish three different nonspecific binding modes for both HUαβ (Koh et al., 2008) and IHF. Both the NAP:DNA mole ratio ([NAP]/[DNA]) and DNA length dictate the dominant NAP binding mode. On sufficiently long DNA, at low [NAP]/[DNA], both HU and IHF populate a noncooperative 34 bp binding mode with a binding constant of ∼ 107 M−1 at 0.082 M Na+. With increasing [NAP]/[DNA], both HU and IHF bound in the noncooperative 34 bp mode progressively convert to two moderately cooperative modes with site sizes of 10 bp and 6 bp and smaller binding constants. As DNA length increases at low [NAP]/[DNA], fractional population of the 34 bp mode increases. The 34 bp mode of IHF exhibits a large negative ΔCp,obs° and an exothermic ΔHobs° (15 - 25°C), similar to previous observations for the specific complex of IHF and wrapped H'-DNA, whereas a small positive ΔCp,obs° and an endothermic ΔHobs° were observed for the 34 bp mode of HU. From these and parallel studies at various salt concentrations we propose that DNA is wrapped on the body of IHF in the nonspecific 34 bp mode like the specific complex of IHF and H'-DNA, whereas DNA is bent but not wrapped in the 34 bp nonspecific HU-DNA complex. Other structural features of the binding modes of HU and IHF deduced from these studies are also discussed.This work was supported by NIH grant GM 23467.