Figure 3.

Synthetic substrate specificity of Atg4B. (A) The positional scanning strategy is depicted for using combinatorial peptide library to define optimal substrate sequence for Atg4B. (B) Summary of cleavage of fluorogenic tetrapeptides is presented. The enzyme concentration was 6–8 µM. ACC production was monitored with assay times varying from 15–60 min. Standard deviation for each measurement shown is <20%. The x-axis indicates the amino-acid tested at the P2, P3 or P4 positions (standard single letter code for natural L-amino acids; O, nor-leucine, hC, cyclohexylalanine, hP, homo-phenylalanine). The y-axis represents the average relative activity expressed as a percent of the best amino acid. ACC fluorescence was monitored using an fmax multiwell fluorescence plate reader at excitation wavelength of 355 nm and an emission wavelength of 460 nm for 15–60 mins.