Rapid identification of phosphopeptide ligands for SH2 domains. Screening of peptide libraries by fluorescence-activated bead sorting

• Published in: The Journal of biological chemistry Vol. 271; no. 28; pp. 16500 - 16505
• Main Authors: Müller, K, Gombert, F O, Manning, U, Grossmüller, F, Graff, P, Zaegel, H, Zuber, J F, Freuler, F, Tschopp, C, Baumann, G
• Format: Journal Article
• Language: English
• Published: United States 12.07.1996
• Subjects: Amino Acid Sequence; Base Sequence; Cell Line, Transformed; DNA, Complementary; Enzyme Precursors - chemistry; Enzyme Precursors - metabolism; Flow Cytometry; Fluorescein-5-isothiocyanate; Fluorescence; Humans; Intracellular Signaling Peptides and Proteins; Ligands; Molecular Sequence Data; Phosphopeptides - chemistry; Phosphopeptides - metabolism; Protein Binding; Protein-Tyrosine Kinases - chemistry; Protein-Tyrosine Kinases - metabolism; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; src Homology Domains; Syk Kinase
• DOI: 10.1074/jbc.271.28.16500
• ISSN: 0021-9258

A method for the identification of high-affinity ligands to SH2 domains by fluorescence-activated bead sorting (FABS) was established. Recombinant SH2 domains, expressed as glutathione S-transferase (GST) fusion proteins, were incubated with a phosphotyrosine (Y*)-containing peptide library. 6.4 x 10(5) individual peptides of nine amino acids in length (EPX6Y*X19X7X19X7X6) were each displayed on beads. Phosphopeptide interaction of a given SH2 domain was monitored by binding of fluorescein isothiocyanate-labeled antibodies directed against GST. High-fluorescence beads were isolated by flow cytometric sorting. Subsequent pool sequencing of the selected beads revealed a distinct pattern of phosphotyrosine-containing motifs for each individual SH2 domain: the SH2 domain of the adapter protein Grb2 predominantly selected beads with the sequence Y*ENDP, whereas the C-terminal SH2 domain of the tyrosine kinase Syk selected Y*EELD, each motif representing the most frequently found residues C-terminal to the phosphotyrosine. For deconvolution studies, soluble phosphopeptides comprising variations of the Grb2 motifs were resynthesized and analyzed by surface plasmon resonance.