tailieunhanh - Báo cáo khoa học: Thermodynamic analysis of the unfolding and stability of the dimeric DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant

We have studied the stability of the histone-like, DNA-binding protein HU from the hyperthermophilic eubacteriumThermotoga maritimaand its E34D mutant by differential scanningmicrocalorimetry and CDunder acidic conditions at various concentrations within the range of 2–225lMof monomer. The thermal unfolding of both proteins is highly reversible and clearly follows a two-state dissociation/unfolding model from the folded, dimeric state to the unfolded, monomericone. | Eur. J. Biochem. 271 1497-1507 2004 FEBS 2004 doi Thermodynamic analysis of the unfolding and stability of the dimeric DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant Javier Ruiz-Sanz1 Vladimir V. Filimonov1 2 Evangelos Christodoulou3 Constantinos E. Vorgias3 and Pedro L. Mateo1 1 Department of Physical Chemistry Faculty of Sciences and Institute of Biotechnology University of Granada Spain 2Institute of Protein Research Russian Academy of Sciences Pushchino Moscow Russia 3Faculty of Biology Department of Biochemistry and Molecular Biology National and Kapodistrian University of Athens Greece We have studied the stability of the histone-like DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant by differential scanning microcalorimetry and CD under acidic conditions at various concentrations within the range of 2-225 M of monomer. The thermal unfolding of both proteins is highly reversible and clearly follows a two-state dissociation unfolding model from the folded dimeric state to the unfolded monomericone. The unfolding enthalpy is very low even when taking into account that the two disordered DNA-binding arms probably do not contribute to the cooperative unfolding whereas the quite small value for the unfolding heat capacity change kJ-K-1-mol-1 stabilizes the protein within a broad temperature range as shown by the stability curves Gibbs energy functions vs. temperature even though the Gibbs energy of unfolding is not very high either. The protein is stable at pH and but becomes considerably less so at pH and below to the point that a simple decrease in concentration will lead to unfolding of both the wild-type and the mutant protein at pH and low temperatures. This indicates that various acid residues lose their charges leaving uncompensated positively charged clusters. The wild-type protein is more .