Dimeric procaspase-3 unfolds via a four-state equilibrium process.
We have examined the folding and assembly of a catalytically inactive mutant 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. However, the midpoint of the second transition as well as the amplitude of the 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 intermediate dissociates to a monomeric intermediate, which then unfolds. By fitting the four-state model to the experimental data, we have determined the free energy change for the first step of unfolding to be 8.3 +/- 1.3 kcal/mol. The free energy change for the dissociation of the dimeric folding intermediate to two monomeric intermediates is 10.5 +/- 1 kcal/mol. The third step in the unfolding mechanism represents the complete unfolding of the monomeric intermediate, with a free energy change of 7.0 +/- 0.5 kcal/mol. These results show two important points. First, dimerization of procaspase-3 occurs as a result of the association of two monomeric folding intermediates, demonstrating that procaspase-3 dimerization is a folding event. Second, the stability of the dimer contributes significantly to the conformational free energy of the protein (18.8 of 25.8 kcal/mol).
Bose
K
K
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695, USA.
Clark
A C
AC
D053148:Caspase 3; D020169:Caspases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D019281:Dimerization; D004792:Enzyme Precursors / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D009154:Mutation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea / Q000494:pharmacology
D004792:Enzyme Precursors; D014508:Urea; D053148:Caspase 3; D020169:Caspases