Polychrome activity profiling distinguishes Cys proteases and their isoforms in plants
Activity-based profiling with fluorescent probes is a powerful tool for the functional characterization of whole enzyme classes in crude proteomes. Here, we discovered that probe cocktails consisting of the E-64 warhead carrying different fluorophores via short linkers distinguishes the labeling of papain-like cysteine proteases and their isoforms because of their differential affinity to these probes. This causes polychrome labeling with specific apparent colors for different protease families and isoforms. Polychrome labeling revealed differential labeling of isoforms of RD21-like proteases carrying C-terminal granulin domains. Molecular modeling of the RD21 isoforms revealed that the C-terminal granulin may create a fluorophore-binding pocket with the substrate-binding groove, implying that the granulin domain influences substrate selectivity. The concept of polychrome labeling may be widely applicable to other chemical probes for the characterization of protein families in all kingdoms of life.