Thermodynamics of DNA Binding and Distortion by the Hyperthermophile Chromatin Protein Sac7d

Sac7d is a hyperthermophile chromatin protein which binds non-specifically to the minor groove of duplex DNA and induces a sharp kink of 66° with intercalation of valine and methionine side-chains. We have utilized the thermal stability of Sac7d and the lack of sequence specificity to define the thermodynamics of DNA binding over a wide temperature range. The binding affinity for poly(dGdC) was moderate at 25 °C image and increased by nearly an order of magnitude from 10 °C to 80 °C. The enthalpy of binding was unfavorable at 25 °C, and decreased linearly from 5 °C to 60 °C. A positive binding heat at 25 °C is attributed in part to the energy of distorting DNA, and ensures that the temperature of maximal binding affinity (75.1±5.6 °C) is near the growth temperature of Sulfolobus acidocaldarius. Truncation of the two intercalating residues to alanine led to a decreased ability to bend and unwind DNA at 25 °C with a small decrease in binding affinity. The energy gained from intercalation is slightly greater than the free energy penalty of bending duplex DNA. Surprisingly, reduced distortion from the double alanine substitution did not lead to a significant decrease in the heat of binding at 25 °C. In addition, an anomalous positive ΔCp of binding was observed for the double alanine mutant protein which could not be explained by the change in polar and apolar accessible surface areas. Both the larger than expected binding enthalpy and the positive heat capacity can be explained by a temperature dependent structural transition in the protein–DNA complex with a Tm of 15–20 °C and a ΔH of 15 kcal/mol. Data are discussed which indicate that the endothermic transition in the complex is consistent with DNA distortion.