Cold denaturation and heat denaturation of Streptomyces subtilisin inhibitor. 1. CD and DSC studies

Cold denaturation and heat denaturation of the protein Streptomyces subtilisin inhibitor (SSI) were studied in the pH range 1.84-3.21 and in the temperature range -3-70 degrees C by circular dichroism and scanning microcalorimetry. The native structure of the protein was apparently most stabilized at about 20 degrees C and was denatured upon heating and cooling from this temperature. Each denaturation was reversible and cooperative, proceeding in two-state transitions, that is, from the native state to the cold-denatured state or from the native state to the heat-denatured state. The two denatured states, however, were not perfect random-coiled structures, and they differed from each other, indicating that there exist three states in this temperature range, i.e., cold denatured, native, and heat denatured. The difference between the cold and heat denaturations was indicated first by circular dichroism. The isodichroic point for the transition from the native state to the cold-denatured state was different from that from the native state to the heat-denatured state in the pH range between 3.21 and 2.45. Moreover, molar ellipticity for the cold-denatured state was different from that of the heat-denatured state, and the transition from the former to the latter was observed at pH values below 2. Values of van't Hoff enthalpies from the native state to the heat-denatured state at pH values between 3.21 and 2.45 were obtained by curve fitting of the CD data, and delta Cp = 1.82 (+/- 0.11) [kcal/(mol.K)] was obtained from the linear plot of the enthalpies against temperature. The parameters obtained from the heat denaturation studies gave curves for delta G zero which were not in agreement with the experimental data in the cold denaturation region when extrapolated to the low temperature. Moreover, the value of the apparent delta Cp for the cold denaturation in the pH range 3.03-2.45 was estimated to be different from that for the heat denaturation, indicating that the mechanism of the cold denaturation of SSI is different from a simple cold denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)