PROBING THE CHEMICAL BASIS OF BINDING-ACTIVITY IN AN SH3 DOMAIN BY PROTEIN SIGNATURE ANALYSIS

Background: Modifying the covalent structure of a protein is an effect ive empirical route to probing three-dimensional structure and biologi cal function. Here we describe a combinatorial protein chemistry strat egy for studying structure-activity relationships in proteins. Our app roach (termed 'protein signature analysis') involves functional select ion from an array of self-encoded protein analogs prepared by total sy nthesis, coupled to a simple chemical readout that unambiguously ident ifies the modified proteins in the resulting active and inactive popul ations. Results: Protein signature analysis was used to study the inte raction of the amino-terminal SH3 domain from the cellular adaptor pro tein c-Crk with its cognate proline-rich peptide, C3G. Using a functio nal selection assay, the qualitative effects of scanning a series of s ynthetic analog units through the amino-acid sequence of the SH3 domai n were evaluated. The analog units were designed to alter both amino-a cid sidechains and the polypeptide backbone within the protein. These chemical studies revealed that the sidechain of Asp150 in the SH3 doma in is essential for ligand binding and that changes in the structure o f the polypeptide backbone can also result in loss of binding activity . Conclusions: These chemical studies have provided new insight into h ow ligand binding is related to the covalent structure of the SH3 doma in. Protein signature analysis is a powerful and conceptually novel wa y of studying the molecular and chemical basis of protein function; it combines the advantages of systematic modification of a protein's che mical structure with the practical convenience of combinatorial synthe sis.