Amino acid frequencies at the substrate positions across the original library, sequences with >1,000-fold enrichment throughout the selection process, and selected candidates. The positions of the substrate sequence are denoted P3, P2, P1, P1′, P2′, and P3′, corresponding to the substrate residues surrounding the protease cleavage site. The frequency of each amino acid at these positions was determined by deep sequencing. The left image displays the amino acid distribution of the original library, the middle image illustrates the distribution of the most enriched sequences, and the right image presents the distribution of the selected candidates. The heatmap is color coded, with red indicating high amino acid frequency at a given position and white indicating low frequency.
