Removal of the pro-domain does not affect the conformation of the procaspase-3 dimer

We have investigated the oligomeric properties of procaspase-3 and a mutant that lacks the pro-domain (called pro-less variant). In addition, we have examined the interactions of the 28 amino acid pro-peptide when added in tr ans to the pro-less variant. By sedimentation equilibrium studies, we have found that procapase-3 is a stable dimer in solution at 25 degreesC and pH 7.2, and we estimate an upper limit for the equilibrium dissociation consta nt of similar to 50 nM. Considering the expression levels of caspase-3 in J urkat cells, we predict that procaspase-3 exists as a dimer in vivo. The pr o-less variant is also a dimer, with little apparent change in the equilibr ium dissociation constant. Thus, in contrast with the long pro-domain caspa ses, the pro-peptide of caspase-3 does not appear to be involved in dimeriz ation. Results from circular dichroism, fluorescence anisotropy, and FTIR s tudies demonstrate that the pro-domain interacts weakly with the pro-less v ariant. The data suggest that the pro-peptide adopts a beta -structure when in contact with the protein, but it is a random coil when free in solution . In addition, when added in trans, the pro-peptide does not inhibit the ac tivity of the mature caspase-3 heterotetramer. On the other hand, the activ e caspase-3 does not efficiently hydrolyze the pro-domain at the NSVD9 sequ ence as occurs when the pro-peptide is in cis to the protease domain. Based on these results, we propose a model for maturation of the procaspase-3 di mer.