Carbonic anhydrase is a globular single-domain protein; its native state is dominated by β-structure. In this work the study of pH-induced equilibrium unfolding of carbonic anhydrase apoform has been carried out. It was shown that the protein denaturation by low pH represents the tree-state transition N → I1 → I2. Addition of urea to I2 leads to its unfolding to the unfolded state I2 → U. Using approximation of pH-induced and urea-induced equilibrium unfolding transition curves the estimation of the stability of carbonic anhydrase native and both intermediate states was carried out. Described approach can be used further for investigation of the influence of amino acid substitutions on the folding process of this protein. Moreover in this work the structural features of carbonic anhydrase conformational states were investigated, obtained results concluded that I1 forms associates and contains nonnative α-helical regions.
carbonic anhydrase, protein folding, intermediate state, equilibrium unfolding, circular dichroism
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