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Thermodynamic analysis of the unfolding and stability of the dimeric DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant.
Eur J Biochem. 2004 Apr; 271(8):1497-507.EJ

Abstract

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 micro 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, monomeric one. 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 (3.7 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 4.00 and 3.75, but becomes considerably less so at pH 3.50 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 3.50 and low temperatures. This indicates that various acid residues lose their charges leaving uncompensated positively charged clusters. The wild-type protein is more stable than its E34D mutant, particularly at pH 4.00 and 3.75 although less so at 3.50 (1.8, 1.6 and 0.6 kJ.mol(-1) at 25 degrees C for DeltaDeltaG at pH 4.00, 3.75 and 3.50, respectively), which seems to be related to the effect of a salt bridge between E34 and K13.

Authors+Show Affiliations

Department of Physical Chemistry, Faculty of Sciences, University of Granada, 18071 Granada, Spain.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15066175

Citation

Ruiz-Sanz, Javier, et al. "Thermodynamic Analysis of the Unfolding and Stability of the Dimeric DNA-binding Protein HU From the Hyperthermophilic Eubacterium Thermotoga Maritima and Its E34D Mutant." European Journal of Biochemistry, vol. 271, no. 8, 2004, pp. 1497-507.
Ruiz-Sanz J, Filimonov VV, Christodoulou E, et al. Thermodynamic analysis of the unfolding and stability of the dimeric DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant. Eur J Biochem. 2004;271(8):1497-507.
Ruiz-Sanz, J., Filimonov, V. V., Christodoulou, E., Vorgias, C. E., & Mateo, P. L. (2004). Thermodynamic analysis of the unfolding and stability of the dimeric DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant. European Journal of Biochemistry, 271(8), 1497-507.
Ruiz-Sanz J, et al. Thermodynamic Analysis of the Unfolding and Stability of the Dimeric DNA-binding Protein HU From the Hyperthermophilic Eubacterium Thermotoga Maritima and Its E34D Mutant. Eur J Biochem. 2004;271(8):1497-507. PubMed PMID: 15066175.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Thermodynamic analysis of the unfolding and stability of the dimeric DNA-binding protein HU from the hyperthermophilic eubacterium Thermotoga maritima and its E34D mutant. AU - Ruiz-Sanz,Javier, AU - Filimonov,Vladimir V, AU - Christodoulou,Evangelos, AU - Vorgias,Constantinos E, AU - Mateo,Pedro L, PY - 2004/4/7/pubmed PY - 2004/5/28/medline PY - 2004/4/7/entrez SP - 1497 EP - 507 JF - European journal of biochemistry JO - Eur J Biochem VL - 271 IS - 8 N2 - 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 micro 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, monomeric one. 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 (3.7 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 4.00 and 3.75, but becomes considerably less so at pH 3.50 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 3.50 and low temperatures. This indicates that various acid residues lose their charges leaving uncompensated positively charged clusters. The wild-type protein is more stable than its E34D mutant, particularly at pH 4.00 and 3.75 although less so at 3.50 (1.8, 1.6 and 0.6 kJ.mol(-1) at 25 degrees C for DeltaDeltaG at pH 4.00, 3.75 and 3.50, respectively), which seems to be related to the effect of a salt bridge between E34 and K13. SN - 0014-2956 UR - https://www.unboundmedicine.com/medline/citation/15066175/Thermodynamic_analysis_of_the_unfolding_and_stability_of_the_dimeric_DNA_binding_protein_HU_from_the_hyperthermophilic_eubacterium_Thermotoga_maritima_and_its_E34D_mutant_ L2 - https://doi.org/10.1111/j.1432-1033.2004.04057.x DB - PRIME DP - Unbound Medicine ER -