Dimeric Procaspase-3 Unfolds via a Four-State Equilibrium Process

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• 作者：Kakoli Bose and A. Clay Clark
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：November 27, 2001
• 年：2001
• 卷：40
• 期：47
• 页码：14236 - 14242
• 全文大小：88K
• 年卷期：v.40,no.47(November 27, 2001)
• ISSN：1520-4995

文摘

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 especiallycaspase-3, is integral to apoptosis. The equilibrium unfolding data demonstrate a plateau between 3 and5 M urea, consistent with an apparent three-state unfolding process. However, the midpoint of the secondtransition as well as the amplitude of the plateau are dependent on the protein concentration. Overall, thedata are well described by a four-state equilibrium model in which the native dimer undergoes anisomeration 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 freeenergy change for the first step of unfolding to be 8.3 ± 1.3 kcal/mol. The free energy change for thedissociation 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, dimerizationof procaspase-3 occurs as a result of the association of two monomeric folding intermediates, demonstratingthat procaspase-3 dimerization is a folding event. Second, the stability of the dimer contributes significantlyto the conformational free energy of the protein (18.8 of 25.8 kcal/mol).