Probing the chemical basis of binding activity in an SH3 domain by protein signature analysis

Tom W. Muir, Philip E. Dawson, Michael C. Fitzgerald, Stephen B.H. Kent

Research output: Contribution to journalArticlepeer-review

14 Scopus citations


Background: Modifying the covalent structure of a protein is an effective empirical route to probing three-dimensional structure and biological function. Here we describe a combinatorial protein chemistry strategy for studying structure-activity relationships in proteins. Our approach (termed 'protein signature analysis') involves functional selection from an array of self-encoded protein analogs prepared by total synthesis, coupled to a simple chemical readout that unambiguously identifies the modified proteins in the resulting active and inactive populations. Results: Protein signature analysis was used to study the interaction of the amino- terminal SH3 domain from the cellular adaptor protein c-Crk with its cognate proline-rich peptide, C3G. Using a functional selection assay, the qualitative effects of scanning a series of synthetic analog units through the amino-acid sequence of the SH3 domain were evaluated. The analog units were designed to alter both amino-acid sidechains and the polypeptide backbone within the protein. These chemical studies revealed that the sidechain of Asp150 in the SH3 domain is essential for ligand binding and that changes in the structure of the polypeptide backbone can also result in loss of binding activity. Conclusions: These chemical studies have provided new insight into how ligand binding is related to the covalent structure of the SH3 domain. Protein signature analysis is a powerful and conceptually novel way of studying the molecular and chemical basis of protein function; it combines the advantages of systematic modification of a protein's chemical structure with the practical convenience of combinatorial synthesis.

Original languageEnglish (US)
Pages (from-to)817-825
Number of pages9
JournalChemistry and Biology
Issue number10
StatePublished - Oct 1996
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Drug Discovery
  • Molecular Medicine
  • Molecular Biology
  • Biochemistry
  • Clinical Biochemistry
  • Pharmacology


  • combinatorial protein chemistry
  • protein array
  • protein signature analysis


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