Dimeric procaspase-3 unfolds via a four-state equilibrium process

We have examined the folding and assembly of a catalytically inactive mutan t of procaspase-3, a homodimeric protein that belongs to the caspase family of proteases. The caspase family, and especially caspase-3, is integral to apoptosis. The equilibrium unfolding data demonstrate a plateau between 3 and 5 M urea, consistent with an apparent three-state unfolding process. Ho wever, the midpoint of the second transition as well as the amplitude of th e plateau are dependent on the protein concentration. Overall, the data are well described by a four-state equilibrium model in which the native dimer undergoes an isomeration to a dimeric intermediate, and the dimeric interm ediate dissociates to a monomeric intermediate, which then unfolds. By fitt ing the four-state model to the experimental data, we have determined the f ree energy change for the first step of unfolding to be 8.3 +/- 1.3 kcal/mo l. The free energy change for the dissociation of the dimeric folding inter mediate to two monomeric intermediates is 10.5 +/- 1 kcal/mol. The third st ep in the unfolding mechanism represents the complete unfolding of the mono meric intemiediate, with a free energy change of 7.0 +/- 0.5 kcal/mol. Thes e results show two important points. First, dimerization of procaspase-3 oc curs as a result of the association of two monomeric folding intermediates, demonstrating that procaspase-3 dimerization is a folding event. Second, t he stability of the dimer contributes significantly to the conformational. free energy of the protein (18.8 of 25.8 kcal/mol).