Figure 7. Expansion of peptide display libraries using Amber suppression.

(A) Non-canonical amino acids used in this study. CMF = 4-carboxymethyl phenylalanine, AzF = 4-azido phenylalanine, and AcK = N-ε-acetyl-L-lysine. (B) Amber suppression in the strep-tagged X₅-Y-X₅ library using CMF. Library surface-display level was monitored by flow cytometry using a fluorophore-labeled StrepMAB antibody for samples with or without Amber suppression components. (C) AzF labeling on bacterial cells using a DIBO-conjugated fluorophore. Cells expressing the X₅-Y-X₅ library, with and without various Amber suppression components, were treated with DIBO-conjugated Alexa Fluor 555 then analyzed by flow cytometry. (D) Heatmaps depicting the specificities of c-Src, Hck, and c-Abl after CMF or acetyl lysine incorporation. Only sequences with one stop codon were used in this analysis. Enrichment scores were log₂-transformed and are displayed on a color scale from blue (disfavored), to white (neutral), to red (favored). Values in heatmaps are the average of three replicates.

Figure 7—figure supplement 1. Stop codon enrichment levels in c-Src X₅-Y-X₅ screens using different analysis methods.

Error bars represent the standard deviations from three screens.

Figure 7—figure supplement 2. Comparison of position-specific enrichments in screens with Amber suppression analyzed in two different ways.

In each plot, the enrichment of specific amino acids or a stop codon, after phosphorylation by c-Src and bead-based selection, were calculated using two different methods. X-values indicate log-transformed enrichment values calculated across all sequences in the library. Y-values indicate log-transformed enrichment values only for sequences that contain exactly one Amber stop codon. The orange-red points correspond to the Amber codon enrichments at all 10 positions, which selectively fall off of the x=y diagonal line.

Figure 7—figure supplement 3. Phosphorylation kinetics of Lys- and AcK-containing consensus peptides against c-Src and c-Abl.

Initial rates measured for each kinase were normalized to the rate of the corresponding cognate consensus peptide. Peptides were used at a concentration of 100 or 20 μM, and the kinases were used at a concentration of 10–50 nM. Error bars represent the standard deviation from three measurements.