Conformational stability of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis, and of the four HBsu variants [F29W], [F47W], [F50W] and [F79W]

Abstract

From denaturation studies with urea a free energy ΔG_u ^H2O of unfolding of 49.8 kJ · mol⁻¹ at 25C was calculated for the histone-like DNA-binding protein HBsu from Bacillus subtilis. Unfolding was monitored by circular dichroism measurements observing the changes of the molar mean residue ellipticity [©] at 222 nm. For the calculation of ΔG_u a two-state model of unfolding, i.e. the unfolding of native dimers into unfolded monomers, was applied. The validity of this model in high ionic strength buffer was proven by measurements at different protein concentrations yielding the same ΔG_u values. Four HBsu variants, each carrying one single point mutation ([F29W], [F47W], [F50W] and [F79W]) were analysed with respect to their stability against unfolding at increasing temperatures and urea concentrations. The ΔG_u values of mutants were calculated using the two-state model and show a reduced stability of the variants [F29W], [F47W], [F50W] and [F79W] in comparison to the wild type HBsu with ΔΔGu values of -9.2 kJ·mol⁻¹, -7.5 kJ·mol⁻¹, -5.9 kJ· mol⁻¹, and -7.5 kJ · mol⁻¹, respectively. Similar Δ ΔG_u values were obtained for the HBsu mutant proteins by thermal unfolding experiments.