A PDZ Domain-Based Detection System for Enzymatic Assays

✍ Scribed by Marc Ferrer; Aaron C. Hamilton; James Inglese

• Publisher: Elsevier Science
• Year: 2002
• Tongue: English
• Weight: 396 KB
• Volume: 301
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.2001.5497

✦ Synopsis

A time-resolved fluorescence resonance energy transfer (TR-FRET) detection method based on the formation of a PDZ domain ⅐ 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 Eu 3؉ chelate-labeled GST-PDZ ([Eu 3؉ ]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.