Thermodynamics and kinetics of unfolding of the thermostable trimeric adenylate kinase from the archaeon Sulfolobus acidocaldarius.

ABSTRACT

The thermal stability of adenylate kinase from the thermoacidophilic archaeon Sulfolobus acidocaldarius was characterized comprehensively using denaturant-induced unfolding, differential scanning calorimetry, circular dichroism spectroscopy, and enzymological inactivation studies. The thermally induced unfolding of the protein is irreversible due to aggregation, whereas the unfolding induced by guanidinium chloride is reversible. The protein is known to be a homotrimer in its native state and we established that it unfolds upon dissociation in the case of denaturant unfolding. We measured the thermodynamic stability of the protein in a temperature range from 5 to 70 degrees C using denaturant unfolding. The protein has a maximum of stability (intrinsic free energy) of 31 kcal/mol-trimer (130 kJ/mol-trimer) at 32 degrees C (based on the linear extrapolation model). The heat capacity change upon unfolding DeltaCp and the m-value were considered to be constant in this temperature range and calculated to be 2.86 kcal/mol-trimer (11.9 kJ/mol-trimer) and 5.67 kcal/mol-trimer M (23.7 kJ/mol-trimer M), respectively. The influence of trimerization on thermodynamic stability was investigated. The several interrelated aspects of thermal stability such as unfolding kinetics, the temperature-dependence of the free energy, and the concentration and temperature-dependencies of the fraction of denatured protein are described quantitatively. The properties of the Gibbs-Helmholtz function of the adenylate kinase from S. acidocaldarius, in particular, and of oligomeric proteins, in general terms, are discussed and compared with the properties of the analogous function for monomeric proteins. Moreover, we discuss methodological aspects we obtained the analytical expression of the denaturant-unfolding isotherm for homotrimeric proteins; we include a formula Appendix containing the derivations of the expressions used.