Fig. 3. Design and synthesis of reactive peptide ligands for covalent PDZΔRGS3 conjugation (PDZΔRGS3 denotes the PDZ domain of PDZ-RGS3). (A) Peptide sequences designed for protein conjugation. (B) Peptide specificity and site selective cysteine conjugation of PDZΔRGS3. PDZΔRGS3 was reacted with different peptides (protein/peptide 20/100 μM, pH 7.4, 1 hour). PDZ mutants were incubated with peptide PD3-I (protein/peptide 20/100 μM, pH 7.4, 12 hours). (C) Reaction kinetics study of peptide PD3-I (protein/peptide 15/75 μM, pH 7.4) for 0 min, 1 min, 10 min, 60 min, 240 min, 480 min and 720 min, respectively. Stoichiometric study of peptide PD3-I from 0.5 equiv. to 10.0 equiv. by incubating with PDZΔRGS3 for 4 hours. (D) Ligand induced protein conjugation. Other proteins containing free Cys would not react with peptide PD3-I (protein/peptide 15/75 μM, pH 7.4, 12 hours). (E) FAM-labelled peptides (PD3-I, 50 μM) and PDZΔRGS3 (10 μg) were incubated with 293 T cell lysates (300 μg) for 24 hours. FL, in-gel fluorescence scanning.
