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A PDZ domain‐based assay for measuring HIV protease activity: Assay design considerations
Author(s) -
Hamilton Aaron C.,
Inglese James,
Ferrer Marc
Publication year - 2003
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.0235603
Subject(s) - pdz domain , peptide , protease , ligand (biochemistry) , enzyme , peptide sequence , biochemistry , chemistry , binding site , biology , gene , receptor
We have recently described a biochemical detection method for peptide products of enzymatic reactions based on the formation of PDZ domain•peptide ligand complexes. The product sensor is based on using masked or cryptic PDZ domain peptide ligands as enzyme substrates. Upon enzymatic processing, a PDZ‐binding motif is exposed, and the product sequence bound specifically by a Eu 3+ chelate‐labeled GST–PDZ ([Eu 3+ ]GST–PDZ). The practical applicability of this PDZ‐based detection method is determined by the affinity of the PDZ domain•peptide ligand interaction, and the efficiency of the enzyme to process the masked peptide ligand. To expand the use of this PDZ‐based detection strategy to a broader range of enzymatic assays, we have taken advantage of the plasticity in ligand recognition by the variety of PDZ domains found in nature. In the original work, the PDZ3 of PSD‐95 was used, which preferentially recognizes the consensus sequence Ser‐X‐Val‐COOH. Here, we show that NHERF PDZ1, which binds to the consensus sequence Thr/Ser‐X‐Leu‐COOH, can be used to extend the flexibility in the recognition of the carboxy‐terminal amino acid of the ligand, and monitor the enzymatic activity of HIV protease. The choices of detection format, for example, TRET or ALPHA, were also investigated and influenced assay design.