Folding and assembly kinetics of procaspase-3.
Caspases are vital to apoptosis and exist in the cell as inactive zymogens. Dimerization is central to procaspase activation because the active sites are comprised of loops from both monomers. Although initiator procaspases are stable monomers until activated on cell death scaffolds, the effector caspases, such as procaspase-3, are stable dimers. The activation mechanisms are reasonably well understood in terms of polypeptide chain cleavage and subsequent active site rearrangements in the dimer, but the mechanisms that govern dimer assembly are not known. To further understand procaspase dimerization, we examined the folding and assembly of procaspase-3 by fluorescence emission, circular dichroism, differential quenching by acrylamide, anisotropy, and enzyme activity assays. Single-mixing stopped-flow refolding studies showed a complex burst phase in which multiple monomeric species form rapidly. At longer times, the monomer folds through several intermediates, some of which appear to be off-pathway or misfolded, before eventually forming a dimerization-competent species. Enzyme activity studies demonstrated a slow rate of dimerization (approximately 70 M(-1) s(-1)). In addition, single-mixing stopped-flow unfolding studies revealed a complex unfolding process with a slow rate of dimer dissociation. Interestingly, multiple dimeric species were observed in the burst phase for unfolding, suggesting that the native ensemble consists of at least two major conformations. Collectively, these results demonstrate complex folding and unfolding behavior for procaspase-3 and suggest that slow dimerization results from the lack of stabilizing native contacts in the initial encounter complex.
Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, North Carolina 27695, USA.
Sara L
SL
Milam
A Clay
AC
Clark
D053148:Caspase 3 / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D019281:Dimerization; D004789:Enzyme Activation; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
D053148:Caspase 3