Folding and assembly kinetics of procaspase-3

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Caspases are key enzymes in apoptosis, existing as inactive zymogens.
• Procaspase-3, an effector caspase, forms stable dimers essential for its activation.
• The mechanisms governing procaspase dimer assembly remain largely unknown.

Purpose of the Study:

• To investigate the folding and assembly mechanisms of procaspase-3.
• To elucidate the kinetics and conformational dynamics of procaspase-3 dimerization.

Main Methods:

• Utilized fluorescence emission, circular dichroism, and anisotropy assays.
• Employed single-mixing stopped-flow refolding and unfolding studies.
• Incorporated differential quenching by acrylamide and enzyme activity assays.

Main Results:

• Procaspase-3 folding exhibits a complex burst phase with rapid formation of multiple monomeric species.
• Monomer folding proceeds through intermediates, including off-pathway or misfolded species, before reaching a dimerization-competent state.
• Dimerization occurs slowly (approx. 70 M(-1) s(-1)), and unfolding reveals multiple dimeric species, indicating at least two native conformations.

Conclusions:

• Procaspase-3 folding and unfolding are complex, non-trivial processes.
• Slow dimerization is attributed to the absence of stabilizing native contacts in the initial encounter complex.
• The native state of procaspase-3 is conformationally dynamic, comprising multiple dimeric forms.