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

The thermal stability of adenylate kinase from the thermoacidophilic archae on Sulfolobus acidocaldarius was characterized comprehensively using denatu rant-induced unfolding, differential scanning calorimetry, circular dichroi sm spectroscopy, and enzymological inactivation studies. The thermally indu ced unfolding of the protein is irreversible due to aggregation, whereas th e unfolding induced by guanidinium chloride is reversible. The protein is k nown to be a homotrimer in its native state and we established that it unfo lds 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 stabilit y (intrinsic free energy) of 31 kcal/mol-trimer (130 kJ/mol-trimer) at 32 d egrees C (based on the linear extrapolation model). The heat capacity chang e upon unfolding Delta C-p and the In-value were considered to be constant in this temperature range and calculated to be 2.86 kcal/mol-trimer (11.9 k J/mol-trimer) and 5.67 kcal/mol-trimer M (23.7 kJ/mol-trimer M), respective ly. The influence of trimerization on thermodynamic stability was investiga ted. The several interrelated aspects of thermal stability such as unfoldin g kinetics, the temperature-dependence of the free energy, and the concentr ation and temperature-dependencies of the fraction of denatured protein are described quantitatively. The properties of the Gibbs-Helmholtz function o f the adenylate kinase from S. acidocaldarius, in particular, and of oligom eric proteins, in general terms, are discussed and compared with the proper ties of the analogous function for monomeric proteins. Moreover, we discuss methodological aspects: we obtained the analytical expression of the denat urant-unfolding isotherm for homotrimeric proteins; we include a formula Ap pendix containing the derivations of the expressions used. (C) 1998 Academi c Press.