title: Fluorescent caged phosphoserine peptides as probes to investigate phosphorylation-dependent protein associations.
abstract: The development of chemical probes for the investigation of the complex phosphorylation signaling cascades that regulate biological events is crucial to understanding these processes. We describe herein a bifunctional probe that enables spatial and temporal release of a biologically active ligand while allowing simultaneous monitoring of its binding to the protein of interest. Substitution of Tyr(-2) for the enviromentally sensitive fluorescent amino acid DANA in the sequence RLYRpSLPA which is known to bind the 14-3-3 protein does not adversely affect binding affinity and allows monitoring of the binding process. The binding of the peptide to 14-3-3 places the fluorescent reporter unit into a hydrophobic pocket, which changes the fluorescent maximum emission intensity and wavelength. At the same time, the newly developed photolabile 1-(2-nitrophenyl)ethyl-caged phosphoserine allows control of the release of the biologically active ligand through unmasking of the key phosphoserine functionality upon UV irradiation.
authors.affiliation: Department of Chemistry and Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
authors.forename: M Eugenio
authors.initials: ME
authors.lastname: Vázquez
authors.forename: Mark
authors.initials: M
authors.lastname: Nitz
authors.forename: Justine
authors.initials: J
authors.lastname: Stehn
authors.forename: Michael B
authors.initials: MB
authors.lastname: Yaffe
authors.forename: Barbara
authors.initials: B
authors.lastname: Imperiali
mesh_terms: D048948:14-3-3 Proteins; D015081:2-Naphthylamine / Q000031:analogs & derivatives* / Q000737:chemistry; D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D009281:Naphthalenes / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014446:Tyrosine 3-Monooxygenase / Q000032:analysis* / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; C471216:6-(2-dimethylaminonaphthoyl) alanine; D005456:Fluorescent Dyes; D009281:Naphthalenes; D010748:Phosphopeptides; D010768:Phosphoserine; C038517:prodan; D015081:2-Naphthylamine; D014446:Tyrosine 3-Monooxygenase; D000409:Alanine