Thermodynamics of protein-DNA interactions

This thesis treats the molecular recognition between DNA-binding proteins and DNA and the way these interactions are affected by the surrounding water solution and dissolved solutes. The work involves studies on the thermodynamics of sequence specific DNA-binding of the human glucocorticoid receptor DNA-binding domain and the nonsequence specific DNA- binding of Sso7d from the archaeon Sulfolobus solfataricus. Several experimental techniques including fluorescence, absorption and nuclear magnetic resonance spectroscopy, gel mobility shift assays and isothermal titration calorimetry have been used in the characterisation of these equilibria.

The glucocorticoid receptor DNA-binding domain binds as a homodimer to sequence specific DNA. We have analysed the effect of biologically relevant DNA base pair alterations in the binding site as well as amino acid mutations in the protein on the thermodynamics of DNA- binding. Comparisons with known structures allow us to rationalise individual differences in measured thermodynamic quantities for the different complexes. For instance, we find that the removal of a thymine methyl at the DNA-protein interface is enthalpically favourable, but entropically unfavourable, which is consistent with a replacement by a water molecule. Furthermore, the studies show that dehydration of the interacting macromolecular surfaces has a large impact on the thermodynamics of complex formation. The sequence specific DNA- binding of the glucocorticoid receptor DNA-binding domain is also affected by salt and pH conditions. A comparison with structural data reveals that a histidine residue at the macromolecular interface is protonated when the protein binds DNA at physiological pH, but only at low ionic strength conditions.

We have shown that the nonsequence specific DNA-binding of Sso7d protects double-stranded DNA from thermal denaturation at low ionic strength conditions. Included is also a more general characterisation of the DNA-binding properties of Sso7d, covering the preferential binding to different polymeric DNA sequences as well as the temperature and pH dependence. We were also able to establish that the salt dependence of the free energy of complex formation is accompanied by a corresponding change in the entropy component, whereas no systematic salt dependence was resolved for the enthalpy of binding. This finding is in agreement with the common view of an entropy dominated salt dependence, but it has not been measured until now for a protein-DNA interaction using calorimetric methods.