Fig. 2. Preferences of apoptotic caspases at the P5 position. (A) The P5 combinatorial library was designed based on general caspase preferences toward acidic amino acid residues at the P1 (Asp) and P3 (Glu) positions. The general architecture of the P5 combinatorial fluorogenic substrate library is Ac-P5-Mix-Glu-Mix-Asp-ACC. (B) Substrate specificity of seven apoptotic caspases at the P5 position. The activity ratio between pentapeptide and the control tetrapeptide substrate lacking a P5 amino acid is shown as a heatmap, where red indicates the best (most preferable) substrates and blue indicates the least preferable substrates. The results are arranged in accordance with caspase 6 preferences. All P5 specificity profiles are presented in the Supplemental Section; “none” – activity toward the corresponding tetrapeptide substrate (Ac-Mix-Glu-Mix-Asp-ACC). (C) The structures of the best amino acid residues for caspase 6 at the P5 position.