Studying receptor-ligand interactions using encoded amino acid scanning

A novel technique is described that allows the synthesis, functional analysis, and quantitative readout of defined arrays of polypeptide analogues in aqueous solution. Key to this approach is the use of a simple encoding- decoding system in which a unique Fmoc-amino acid tag is covalently attached to the C terminus of each member of a molecular array through a selectively cleavable bond. These tags can be cleanly removed from the molecules they encode, allowing single-step characterization and quantification of the entire mixture by HPLC. The utility of this technique is illustrated through the preparation of an array of proline-rich sequences based on the exchange factor C3G, one of the natural ligands of the N-terminal SH3 domain from the proto-oncogene, c-Crk. The array was designed to systematically modify those residues within the C3G peptide ligand thought to make key interactions with the c-Crk SH3 domain. Using competition binding experiments, it was possible to determine the relative ED₅₀ values for the entire array of molecules simultaneously. These studies revealed that in order to maintain optimal binding to the SH3 domain, the P-3 side chain of the ligand must be positively charged and the P-0 side chain must be hydrophobic and extend beyond the γ-carbon. The excellent correlation between these relative ED₅₀ values and a series of relative K(d) values determined from individual peptides suggests that this approach may be useful in determining, in a parallel fashion, the relative biological activities of arrays of polypeptides.