RAPID IDENTIFICATION OF PHOSPHOPEPTIDE LIGANDS FOR SH2 DOMAINS - SCREENING OF PEPTIDE LIBRARIES BY FLUORESCENCE-ACTIVATED BEAD SORTING

A method for the identification of high-affinity ligands to SH2 domain s by fluorescence-activated bead sorting (FABS) was established, Recom binant SH2 domains, expressed as glutathione S-transferase (GST) fusio n proteins, were incubated with a phosphotyrosine (Y) containing pept ide library, 6.4 x 10(5) individual peptides of nine amino acids in le ngth (EPX(6)YX(19)X(7)X(19)X(7)X(6)) were each displayed on beads, Ph osphopeptide interaction of a given SH2 domain was monitored by bindin g of fluorescein isothiocyanate-labeled antibodies directed against GS T. High-fluorescence beads were isolated by flow cytometric sorting, S ubsequent pool sequencing of the selected beads revealed a distinct pa ttern of phosphotyrosine-containing motifs for each individual SH2 dom ain: the SH2 domain of the adapter protein Grb2 predominantly selected beads with the sequence YENDP, whereas the C-terminal SH2 domain of the tyrosine kinase Syk, selected YEELD, each motif representing the most frequently found residues C-terminal to the phosphotyrosine, For deconvolution studies, soluble phosphopeptides comprising variations o f the Grb2 motifs were resynthesized and analyzed by surface plasmon r esonance.