Stability diagram and unfolding of a modified cytochrome c: what happens in the transformation regime?

We determined the stability diagram of a modified cytochrome c protein in a glycerol water mixture by measuring the first and the second moment of the fluorescence from the chromophore as a function of temperature and pressure. Temperature and pressure were varied between 273 and 363 K and 0.0001 and 1 GPa, respectively. The shift of the fluorescence maximum showed a characteristic sigmoid-like pattern from which information on the microscopic processes during unfolding is obtained: as the transformation regime is entered, the fluorescence shows a significant blue shift. The conclusion is that water molecules get into contact with the chromophore. They lead to strong electrostatic contributions in the solvent shift, which counteract the red shifting dispersion interactions. Assuming that there are just two relevant states that determine the stability diagram, the complete set of thermodynamic parameters can be determined from the data. However, under certain pressure-temperature conditions the fluorescence pattern is more complicated, pointing toward reentrant transitions and, possibly, to consecutive steps in the unfolding process.