title: Semisynthesis of unnatural amino acid mutants of paxillin: protein probes for cell migration studies.
abstract: Caged phosphopeptides and phosphoproteins are valuable tools for dissecting the dynamic role of phosphorylation in complex signaling networks with temporal and spatial control. Demonstrating the broad scope of phosphoamino acid caging for studying signaling events, we report here the semisynthesis of a photolabile precursor to the cellular migration protein paxillin, which is a complex, multidomain phosphoprotein. This semisynthetic construct provides a powerful probe for investigating the influence that phosphorylation of paxillin at a single site has on cellular migration. The 61-kDa paxillin construct was assembled using native chemical ligation to install a caged phosphotyrosine residue at position 31 of the 557-residue protein, and the probe includes all other binding and localization determinants in the paxillin macromolecule, which are essential for creating a native environment to investigate phosphorylation. Following semisynthesis, paxillin variants were characterized through detailed biochemical analyses and by quantitative uncaging studies.
authors.affiliation: Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Vogel
authors.forename: Barbara
authors.initials: B
authors.lastname: Imperiali
mesh_terms: D019943:Amino Acid Substitution; D002465:Cell Movement; D009154:Mutation; D051419:Paxillin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry; D019000:Phosphotyrosine / Q000235:genetics*; D011485:Protein Binding; D015202:Protein Engineering; D011498:Protein Precursors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D051419:Paxillin; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D010770:Phosphotransferases