The thermal unfolding of the C85S/C152E double mutant of dihydrofolate reductase from E. coli has been investigated using differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. Both calorimetric and spectroscopic data reveal a significant concentration dependence of the melting profiles, even in the range of low concentrations (below 20 M) where the temperature induced unfolding is fully reversible. At pH 7.0, the temperature dependence of the partial molar heat capacity presents two peaks, the main one positioned at 40 C and a small high temperature peak centered at 93 C. Increasing the concentration of protein shifts the first transition to lower temperatures and the second one to higher temperatures. Thermodynamic analysis of the heat capacity function at different concentrations suggests the presence of a significantly populated intermediate state involved in a reversible association process. The heat capacity and the enthalpy change occurring at the first transition are relatively small in comparison with proteins of similar molecular weight (i.e., Cp = 1200 cal/mol?K and H = 65 kcal/mol at a protein concentration of ~ 1 mg/ml). In accordance with this interpretation, the second peak at high temperature would reflect the dissociation of the oligomer formed by the partially folded state into the unfolded monomers. To confirm the proposed model, limited proteolysis experiments have been performed at 25 and 45 C in order to detect the presence of a stable fragment responsible for the association process occurring during the unfolding to the intermediate state. This fragment has been identified at 45 C as a ~ 10 kDa chain of the major domain, the secondary structure of which is predominantly b-sheet. This result is in good agreement with prediction of the most probable partially folded state based on the structural parametrization.
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