Heat denaturation and cold denaturation of Escherichia coli RNase HI investigated by circular dichroism

Circular dichroism has been used to investigate the thermodynamics of the thermal unfolding of Escherichia coli ribonuclease HI as a function of pH over the pH range 0–4. This protein undergoes a reversible thermal conformational change from the native state to the denatured state with an isodichroic point. The calculated thermodynamic values at pH 3.0 are as follows: tm = 50.2°C, ΔHm (tm) = 93.8 kcal mol−1, ΔG (25°) = 6.08 kcal mol−1, and ΔG (10°C) = 8.14 kcal mol−1. At pH 4, ΔG (25°C) = 10 kcal mol−1 and ΔG (10°C) = 12 kcal mol−1. At a pH below 2, this protein denatures at 25°C with ΔG (25°C) = −1 kcal mol−1, but it is stable at 10°C with ΔG (10°C) = 2 kcal mol−1. The ΔCp value determined from the ΔHm(Tm) versus Tm plot is 1.4 kcal mol−1 K−1. The thermal unfolding curves at pH values above 2.18 showed a highly cooperative thermal transition. Cold denaturation was observed at temperatures below 10°C between pH 2.03 and 1.55. A cooperative heat denaturation was also observed over this pH range. At pH values lower than 1.45, cold denaturation was not observed. A broad thermal transition was observed between pH 0.83 and 0.53.