Frank Otto Gombert - Rapid Identification of Phosphopeptide Ligands for SH2 Domains

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      Publication Details (including relevant citation   information):

      Müller, K.; Gombert, F. O.; Manning, U.; Grossmüller, F.; Graff,   P.; Zaegel, H.; Zuber, J. F..; Freuler, F.; Tschopp, C.; Baumann,   G.. Rapid Identification of Phosphopeptide Ligands for SH2   Domains. J. Biol. Chem. 1996,  271, 16500-16505.


      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 +ù 105   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

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