A PDZ domain-based assay for measuring HIV protease activity: assay design considerations.
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.
Hamilton
Aaron C
AC
Department of Automated Biotechnology, North Wales, Pennsylvania 19454, USA.
Inglese
James
J
Ferrer
Marc
M
D017931:DNA Primers / Q000737:chemistry; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005063:Europium; D005453:Fluorescence; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D016333:HIV Protease / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling
D017931:DNA Primers; D008024:Ligands; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005063:Europium; D005982:Glutathione Transferase; D016333:HIV Protease