title: Sequence-based design of a peptide probe for the APC tumor suppressor protein.
abstract: BACKGROUND
  Proteins form specific associations, but predictive rules for protein pairing are generally unknown. Here, we describe amino-acid sequence patterns capable of mediating specific pairing of a widespread protein motif: the parallel, dimeric, alpha-helical coiled coil. The pairing rules were tested by designing a 54-residue peptide (anti-APCp1) that is predicted to dimerize preferentially with a coiled-coil sequence from the adenomatous polyposis coli (APC) tumor suppressor protein.
  
  
  RESULTS
  As judged by circular dichroism, ultracentrifugation and native gel electrophoresis, anti-APCp1 formed a specific, helical, dimeric complex with the target APC coiled coil. On western blots of APC fragments expressed in Escherichia coli, the designed peptide detected a pattern of bands identical to the pattern detected by an antibody directed against the APC coiled coil. Peptide-mediated precipitation experiments showed that anti-APCp1 bound and sequestered wild-type and mutant APC proteins in extracts of human colon cancer cell lines. In addition, binding of the designed peptide preserved native APC-beta-catenin complexes.
  
  
  CONCLUSIONS
  These biochemical experiments demonstrate that the anti-APC peptide preferentially forms a heterodimeric coiled coil with mutant and full-length APC proteins. The specificity of the designed peptide is sufficient to support several applications that commonly use antibodies. The observed specificity of anti-APCp1 validates the pairing rules used as the basis for the probe design, and it suggests that residues in the core positions of coiled coils help impart pairing selectivity.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley 94720-3206, USA.
authors.forename: V A
authors.initials: VA
authors.lastname: Sharma
authors.forename: J
authors.initials: J
authors.lastname: Logan
authors.forename: D S
authors.initials: DS
authors.lastname: King
authors.forename: R
authors.initials: R
authors.lastname: White
authors.forename: T
authors.initials: T
authors.lastname: Alber
mesh_terms: D025601:Adenomatous Polyposis Coli Protein; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002942:Circular Dichroism; D003110:Colonic Neoplasms / Q000737:chemistry; D003598:Cytoskeletal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D015195:Drug Design; D017491:Genes, APC; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D014407:Tumor Cells, Cultured; D014461:Ultracentrifugation
chemical_list: D025601:Adenomatous Polyposis Coli Protein; D003598:Cytoskeletal Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins