Thermostability of proteins: Role of metal binding and pH on the stability of the dinuclear CuA site of Thermus thermophilus
Agnieszka Sujak 1, Nusrat J.M. Sanghamitra 1, Oliver Maneg 2, Bernd Ludwig 2 and Shyamalava Mazumdar 1*
1 Tata Institute of Fundamental Research
2 J. W. Goethe-Universitat
* To whom correspondence should be addressed. E-mail: shyamal{at}tifr.res.in.
Submitted on November 16, 2006
Revised on December 19, 2006
Accepted on 18 June 2007
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Abstract |
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The dinuclear copper center (TtCuA) forming the electron entry site in the subunit II of the cytochrome c oxidase in Thermus thermophilus shows high stability towards thermal as well as denaturant-induced unfolding of the protein at ambient pH. We have studied the effect of pH on the stability of the holo-protein as well as of the apo-protein by UV-visible absorption, far-UV and visible circular dichroism spectroscopy. The results show that the holo- protein both in the native mixed-valence state as well as in the reduced state of the metal ions and the apo- protein of TtCuA were extremely stable towards unfolding by guanidine hydrochloride (GuHCl) at ambient pH. The thermal unfolding studies at different pH suggested that decreasing pH had almost no effect on the thermal stability of the protein in the absence of the denaturant. However, the stability of the proteins in presence of the denaturant was considerably decreased on lowering the pH. Moreover, the stability of the holo-protein in the reduced state of the metal ion was found to be lower than that in the mixed-valence state at the same pH. The denaturant-induced unfolding of the protein at different pH was analysed using a two-state unfolding model. The values of the free energy of unfolding were found to increase with pH. The holoprotein showed that the variation of the unfolding free energy was associated with a pKa of ~5.5. This is consistent with the model that the protonation of a histidine residue may be responsible for the decrease in the stability of the holo-protein at low pH. The results were interpreted in the light of the reported crystal structure of the protein.
Key Words:
Copper protein, CuA center, Thermal unfolding, protein unfolding, thermodynamic stability, thermostability
