A PDZ domain-based detection system for enzymatic assays.
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 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.
Ferrer
Marc
M
Department of Automated Biotechnology, Merck Research Laboratories, 503 Louise Lane, North Wales, Pennsylvania 19454, USA.
Hamilton
Aaron C
AC
Inglese
James
J
D002268:Carboxypeptidases / Q000032:analysis; D004735:Energy Transfer; D005063:Europium; D005453:Fluorescence; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011786:Quality Control; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling; D013379:Substrate Specificity; D062826:Zonula Occludens-1 Protein
D008024:Ligands; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010455:Peptides; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D062826:Zonula Occludens-1 Protein; C033652:postsynaptic density proteins; D005063:Europium; D005982:Glutathione Transferase; D010744:Phosphoric Monoester Hydrolases; D002268:Carboxypeptidases