Studies of irreversible heat denaturation of green fluorescent protein by differential scanning microcalorimetry

Heat denaturation of green fluorescent protein (the GFP-cycle3 mutant) was studied by the method of differential scanning microcalorimetry. Activation energy values for two stages of GFP unfolding were calculated from the calorimetric data using the model of irreversible denaturation. Dependences of activation energy and denaturation enthalpy on the temperature of the maxima of corresponding stages of denaturation were obtained, which allow estimating the corresponding increments of heat capacity. Based on the known correlations of the structure and energy parameters, it was concluded that the first transition state is close to the native state, whereas the second transition state is close to the denatured state, judging by the exposure of hydrophobic groups to the solvent.