title: Mapping protein-protein interactions with phage-displayed combinatorial peptide libraries and alanine scanning.
abstract: One avenue for inferring the function of a protein is to learn what proteins it may bind to in the cell. Among the various methodologies, one way for doing so is to affinity select peptide ligands from a phage-displayed combinatorial peptide library and then to examine if the proteins that carry such peptide sequences interact with the target protein in the cell. With the protocols described in this chapter, a laboratory with skills in microbiology, molecular biology, and protein biochemistry can readily identify peptides in the library that bind selectively, and with micromolar affinity, to a given target protein on the time scale of 2 months. To illustrate this approach, we use a library of bacteriophage M13 particles, which display 12-mer combinatorial peptides, to affinity select different peptide ligands for two different targets, the SH3 domain of the human Lyn protein tyrosine kinase and a segment of the yeast serine/threonine protein kinase Cbk1. The binding properties of the selected peptide ligands are then dissected by sequence alignment, Kunkel mutagenesis, and alanine scanning. Finally, the peptide ligands can be used to predict cellular interacting proteins and serve as the starting point for drug discovery.
authors.affiliation: Department of Biological Sciences, University of Illinois at Chicago, 845 W. Taylor St., Chicago, IL, 60607, USA.
authors.forename: Malgorzata E
authors.initials: ME
authors.lastname: Kokoszka
authors.forename: Brian K
authors.initials: BK
authors.lastname: Kay
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D017104:Bacteriophage M13 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D019151:Peptide Library; D010449:Peptide Mapping / Q000379:methods*; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D019151:Peptide Library; D029701:Saccharomyces cerevisiae Proteins; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; C423362:CBK1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D000409:Alanine