A PDZ Domain-Based Detection System for Enzymatic Assays

Abstract

A time-resolved fluorescence resonance energy transfer (TR-FRET) detection method based on the formation of a PDZ domain · peptide ligand complex has been developed for enzymatic assays as an alternative to immuno-based detection strategies. The enzyme substrate is a “masked” biotinylated PDZ domain peptide ligand containing the consensus sequence Ser–X–Val-COOH. The critical residues in the binding consensus sequence of the ligand have been modified, for example, by phosphorylation of Ser or C-terminal extensions, providing binding-incompetent PDZ domain peptides. On processing by the corresponding enzyme, the binding epitope is exposed, and the product sequence is recognized specifically by Eu3+ chelate-labeled GST–PDZ ([Eu3+]GST–PDZ) (GST–PDZ-glutathione S-transferase fused to PDZ domain). A ternary complex is subsequently formed by addition of allophycocyanin-labeled streptavidin ([XL665]SA), which binds to the biotinylated N terminus of the peptide, and detected by TR-FRET. Reported here are examples of the applicability of this detection strategy to three enzymatic systems, an endoprotease, an exoprotease, and a Ser/Thr phosphatase.