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Structural and biochemical characterization of a fluorogenic rhodamine-labeled malarial protease substrate
Michael J Blackman1, John E T Corrie, John C Croney
1National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. mblackm@nimr.mrc.ac.uk
Biochemistry
|October 3, 2002
Summary
A novel fluorescence assay was developed to study the malarial protease PfSUB-1. This assay uses labeled peptides that become fluorescent upon cleavage, enabling high-throughput screening for inhibitors.
Area of Science:
- Biochemistry
- Parasitology
- Biophysics
Background:
- The malarial protease Plasmodium falciparum subtilisin-like protease 1 (PfSUB-1) is crucial for parasite development.
- Activation of PfSUB-1 involves autocatalytic cleavage at a specific Asp-Asn bond within an internal sequence.
Purpose of the Study:
- To develop a continuous, fluorescence-based assay for measuring PfSUB-1 activity.
- To enable high-throughput screening for inhibitors of PfSUB-1.
Main Methods:
- Synthesis of a decapeptide based on the PfSUB-1 cleavage motif, labeled with iodoacetamidotetramethylrhodamine (IATR).
- Utilizing the fluorescence quenching effect of rhodamine dimer formation in the intact peptide.
- Monitoring fluorescence increase upon peptide cleavage by recombinant PfSUB-1.
Main Results:
- Doubly labeled peptides exhibited low fluorescence due to rhodamine dimerization and self-quenching.
- Cleavage by PfSUB-1 dissociated the rhodamine dimers, leading to a ~30-fold fluorescence increase.
- NMR and time-resolved fluorescence confirmed rhodamine dimer structure and dynamics, indicating an equilibrium between open and closed forms.
Conclusions:
- A novel, sensitive fluorescence assay for PfSUB-1 activity was established.
- The assay allows for quantitative kinetic measurements and high-throughput screening for PfSUB-1 inhibitors.
- Understanding rhodamine dimer behavior is key to the assay's mechanism.