Fig. 3.

Caspase-6 displays variable conformational flexibility across the states of proteolytic activation. Differences in deuterium uptake (daltons) of the corresponding peptic peptides identified in caspase-6 maturation variants [(A) D23A D179A, (B) D23A D179CT, (C) ΔN D179CT, and (D) ΔN D179CT + VEID)] compared with the zymogen (FL C163S) at the indicated time points of exposure to deuterated solvent. The residue numbers for each peptic peptide are listed with the corresponding secondary structural elements. The asterisk designates the D to A substitution at the indicated proteolytic cleavage site, rendering that site uncleavable. These statistically significant differences in the H/D exchange between the caspase-6 maturation variants [(E) D23A D179A, (F) D23A D179CT, (G) ΔN D179CT, and (H) ΔN D179CT + VEID)] and the zymogen (FL C163S) after 2 h of incubation were mapped onto the model structure of caspase-6 shown in both ribbon and surface representations. The asterisk designates D to A mutation in the proteolytic cleavage site in caspase-6. For these data, a deuterium uptake difference greater than 0.6 Da is considered significant at the 98% confidence interval. The intensities of the blue and red colors indicate peptides that undergo either a statistically significant decrease (less exchangeable/flexible) or increase (more exchangeable/flexible), respectively, during H/D exchange along the path of caspase-6 proteolytic activation. (I) Representative deuterium incorporation plots of key peptic peptides identified in caspase-6 maturation variants covering the regions of 26–32, 90s, and 130s; the linker; and the substrate-binding loops L1–L4. The representative MS spectra of the highlighted peptic peptides are shown in Figs. S5 and S6. Error, SD of duplicate H/DX-MS measurements done on 2 separate days.