Figure 4. Monomeric and dimeric states of caspase-9 have different unfolding properties.

Thermal denaturation profile monitored by circular dichroism (left) with the thermal melting temperatures (T_m) for each transition listed and circular dichroism spectra (right) of various forms of caspase-9 (top schematics).

(A) Two melting transitions are observed in the cleaved full-length, monomeric, caspase-9.

(B) and (C) Thermal denaturation profile of cleaved caspase-9 core and the CARD, respectively, showing that the first melting transition in full-length, monomeric caspase-9 (A) is due to the unfolding of the core, while the second is due to that of the CARD.

(D) Full-length, cleaved caspase-9 is dimeric in the presence of an active-site ligand z-VAD-fmk. Upon thermal denaturation, caspase-9 in this state exhibits a single melting transition, likely due either to dimerization, or the presence of an ordered active site, or both.

(E) Dimeric, cleaved caspase-9 ΔCARD with bound z-VAD-fmk is stabilized by 14°C compared to monomeric, cleaved ΔCARD (B).

(F) Constitutive dimer (cDimer) full-length caspase-9 cleaved at the intersubunit linker exhibits two melting transitions, suggesting independent unfolding of CARD and core domains.

(G) The secondary structure of caspase-9 with (+ z-VAD-fmk) and without (FL monomer, cDimer and cDimer C287A) an active-site ligand bound was assessed by circular dichroism spectroscopy. C9 FL cDimer is a constitutive dimer variant of caspase-9. There is no significant change in the helical content of caspase-9 upon dimerization and substrate binding. Estimation of helix content was performed using BeStSel structure prediction and fold recognition software (http://bestsel.elte.hu/index.php) (62).

Plots are representative of three trials. T_m values shown are means ± SEM of three trials done on three separated days.