Effect of Mutation of the Sac7d Intercalating Residues on the Temperature Dependence of DNA Distortion and Binding Thermodynamics

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Abstract
Sac7d is a small chromatin protein from the hyperthermophile Sulfolobus acidocaldarius which kinks duplex DNA by ∼66° at a single base pair step with intercalation of V26 and M29 side chains. Site-directed mutagenesis coupled with calorimetric and spectroscopic data has been used to characterize the influence of the intercalating side chains on the structure and thermodynamics of the DNA complex from 5 to 85 °C. Two single-alanine substitutions (V26A and M29A) and five double-glycine, -alanine, -leucine, -phenylalanine, and -tryptophan substitutions of the surface residues have been created. NMR and fluorescence titrations indicated that the substitutions had little effect on the structure of the protein or DNA binding site size. Each of the mutant proteins demonstrated a temperature-dependent binding enthalpy which was correlated with a similar temperature dependence in the structure of the complex reflected by changes in fluorescence and circular dichroism. A positive heat capacity change (ΔCp) for DNA binding was observed for only those mutants which also demonstrated a thermotropic structural transition in the complex, and the temperature range for the positive ΔCp coincided with that observed for the structural transition. The thermodynamic data are interpreted using a model in which binding is linked to an endothermic distortion of the DNA in the complex. The results support the proposal that the unfavorable enthalpy of binding of Sac7d at 25 °C is due in part to the distortion of DNA.