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A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease.


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.

SUBMITTER: Maus H 

PROVIDER: S-EPMC9793963 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease.

Maus Hannah H   Hinze Gerald G   Hammerschmidt Stefan Josef SJ   Basché Thomas T   Schirmeister Tanja T  

Protein science : a publication of the Protein Society 20230101 1


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 fractio  ...[more]

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2019-03-21 | GSE128625 | GEO