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Authors: Maus, Hannah
Hinze, Gerald
Hammerschmidt, Stefan Josef
Basché, Thomas
Schirmeister, Tanja
Title: A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease
Online publication date: 16-Feb-2024
Year of first publication: 2023
Language: english
Abstract: Ligand binding to proteins often is accompanied by conformational transitions. Here, we describe a competition assay based on single molecule Förster resonance energy transfer (smFRET) to investigate the ligand-induced conformational changes of the dengue virus (DENV) NS2B-NS3 protease, which can adopt at least two different conformations. First, a competitive ligand was used to stabilize the closed conformation of the protease. Subsequent addition of the allosteric inhibitor reduced the fraction of the closed conformation and simultaneously increased the fraction of the open conformation, demonstrating that the allosteric inhibitor stabilizes the open conformation. In addition, the proportions of open and closed conformations at different concentrations of the allosteric inhibitor were used to determine its binding affinity to the protease. The KD value observed is in accordance with the IC50 determined in the fluorometric assay. Our novel approach appears to be a valuable tool to study conformational transitions of other proteases and enzymes.
DDC: 540 Chemie
540 Chemistry and allied sciences
610 Medizin
610 Medical sciences
Institution: Johannes Gutenberg-Universität Mainz
Department: FB 09 Chemie, Pharmazie u. Geowissensch.
Place: Mainz
Version: Published version
Publication type: Zeitschriftenaufsatz
Document type specification: Scientific article
License: CC BY-NC-ND
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Journal: Protein science
Pages or article number: 4526
Publisher: Wiley
Publisher place: Hoboken, NJ
Issue date: 2023
ISSN: 1469-896X
Publisher DOI: 10.1002/pro.4526
Appears in collections:DFG-491381577-H

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