CONFORMATIONAL STABILITY OF THE DNA-BINDING HISTONE-LIKE PROTEIN, HBSU, FROM BACILLUS-SUBTILIS, AND OF THE 4 HBSU VARIANTS [F29W], [F47W], [F50W] AND [F79W]

From denaturation studies with urea a free energy Delta G(u)(H)2(O) of unfolding of 49.8 kJ. mol(-1) at 25C was calculated for the histone-l ike DNA-binding protein HBsu from Bacillus subtilis. Unfolding was mon itored by circular dichroism measurements observing the changes of the molar mean residue ellipticity [Theta] at 222 nm. For the calculation of Delta G(u), a two-state model of unfolding, i.e. the unfolding of native dimers into unfolded monomers, was applied. The validity of thi s model in high ionic strength buffer was proven by measurements at di fferent protein concentrations yielding the same Delta G(u), values. F our HBsu variants, each carrying one single point mutation ([F29W], [F 47W], [F50W] and [F79W]) were analysed with respect to their stability against unfolding at increasing temperatures and urea concentrations. The Delta G(u), values of mutants were calculated using the two-state model and show a reduced stability of the variants [F29W], [F47W], [F 50W] and [F79W] in comparison to the wild type HBsu with Delta Delta G u 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 Delta Delta G(u) values were obta ined for the HBsu mutant proteins by thermal unfolding experiments.