Site-specific DNA binding of architectural protein integration host factor (IHF) is involved in formation of functional multiprotein-DNA assemblies in Escherichia coli, while non-specific binding of IHF and other histone-like proteins serves to structure the nucleoid. Here, we report an isothermal titration calorimetry study of the thermodynamics of binding IHF to a 34 bp fragment composed entirely of the specific H′ site from λ-phage DNA. At low to moderate [K+] (60-100 mM), strong competition is observed between specific and non-specific binding as a result of a low specificity ratio (∼102) and a very small non-specific site size. In this [K+] range, both specific and non-specific binding are enthalpy-driven, with large negative enthalpy, entropy and heat capacity changes and binding constants that are insensitive to [K+]. Above 100 mM K+, only specific binding is observed, and both the binding constant and the magnitudes of enthalpy, entropy and heat capacity changes all decrease strongly with increasing [K+]. When interpreted in the context of the structure of the specific complex, the thermodynamics provide compelling evidence for a previously unrecognized design principle by which proteins that form extensive binding interfaces with nucleic acids control binding constants, binding site sizes and effects of temperature and ion concentrations on stability and specificity. We propose that up to 22 of the 23 IHF cationic side-chains that are located within 6 Å of DNA phosphate oxygen atoms in the complex, are masked in the absence of DNA by pairing with anionic carboxylate groups in intramolecular salt-bridges (dehydrated ion-pairs). These salt-bridges increase in stability with increasing temperature and decreasing [K+]. To explain the unusual thermodynamics of IHF-DNA interactions, we propose that both specific and non-specific binding at low [K+] require disruption of salt-bridges (as many as 18 for specific binding) whereupon many of the unmasked charged groups hydrate and the cationic groups interact with DNA. From structural or thermodynamic parallels with IHF, we propose that large-scale coupling of disruption of protein salt-bridges to DNA binding is significant for other large-interface DNA wrapping proteins including the nucleosome, lac repressor core tetramer, RNA polymerase core protein, HU and SSB.
|Number of pages||23|
|Journal||Journal of Molecular Biology|
|State||Published - Jul 6 2001|
Bibliographical noteFunding Information:
We thank Dr Phoebe Rice (University of Chicago) for her interest in and encouragement of this project, and for the gift of protein for initial experiments with IHF; Dr Craig A. Bingman (Columbia University) for assistance in preparing Figures 8 and 9 ; Dr Darrell McCaslin of the University of Wisconsin-Madison Biophysics Instrument Facility for expert assistance with analytical ultracentrifugation experiments; Mr Mike Capp for assistance with the large-scale preparation of IHF and with DNA purification; Mr Philip Johnson from the Pilot Plant at the University of Wisconsin-Madison for growing 300 l of bacteria; Mr Jeff Ballin for helpful discussions about salt-bridges; and Ms Sheila Aiello for assistance with manuscript preparation. We thank Dr T. M. Lohman (Washington University) and the referees for detailed comments on the manuscript. This research was supported by NIH grant GM23467. J.A.H. acknowledges support from an NIH Molecular Biophysics traineeship.
- Integration host factor
- Isothermal titration calorimetry
- Protein-DNA interactions
ASJC Scopus subject areas
- Structural Biology
- Molecular Biology