Fig. 3: Role of C-terminal extension in structural and functional integrity.
a View of the hsALAS2 active site loop conformation within one monomer (catalytic domain light green, Ct-extension dark green). The active site loop from the open and closed forms of rcALAS (PDB 2BWN) are overlayed onto hsALAS2. b SDS-PAGE of the purified samples from hsALAS2ΔN142 WT (square) and R511E variant (up triangle), as well as hsALAS2ΔN142ΔC545 WT (circle) and R511E variant (down triangle). c Midpoint melting temperatures, determined by differential scanning fluorimetry, of the four samples in panel b. Error bars represent mean values ±SEM from n = 3 technical replicates. **p-values determined by two sample t-test; p = 0.0026, 0.0011, and 0.0002 from left to right. d, e Progression curves for recombinant hsALAS2ΔN142 WT and R511E variant, for both His-tagged and untagged proteins. Specific activity is equivalent to the initial velocity normalized to protein in each assay. Succinyl-CoA and glycine titrations contained 50 mM glycine and 100 μM succinyl-CoA, respectively. Each nonlinear regression line was fitted to data from two biological replicates (n = 2), with the data for each replicate (average of two technical replicates) plotted as distinct symbols (circle and square). Source data for Fig. 3b–e are provided as a Source Data file.
