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.