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Fig. 4
Effect of substitutions at position +5 on binding affinity. (A) Direct binding experiments with various analogues (for the peptide sequences, see Table 1). Substitution of L5 with W caused a dramatic increase in binding affinity, which was partially lost with the additional substitution of D4 with E. The following experimental conditions were used: 0.10 nM CF-P9W5, 0.10 nM CF-P9E4W5, and 1.0 nM CF-P9. Data for CF-P9 are repeated here for comparison. (B) Displacement assay, performed with various analogues (for the peptide sequences, see Table 1). A concentration of labeled peptide equal to 0.10 nM CF-P9W5, interacting with the N-SH2 domain (3.3 nM N-SH2), was displaced with increasing amounts of the unlabeled peptides. The bound fraction of the labeled peptide is reported as a function of the concentration of the competing, unlabeled peptide. The results of independent, replicate experiments (n = 5 for CF-P9 and P8W5, n = 4 for CF-P9W5, and n = 3 for CF-P9E4W5, P8, P8F5, and P8E4W5) are reported with different symbols and were fit collectively.