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http://doi.org/10.25358/openscience-10096| 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 |
| ROR: | https://ror.org/023b0x485 |
| DOI: | http://doi.org/10.25358/openscience-10096 |
| Version: | Published version |
| Publication type: | Zeitschriftenaufsatz |
| Document type specification: | Scientific article |
| License: | CC BY-NC-ND |
| Information on rights of use: | https://creativecommons.org/licenses/by-nc-nd/4.0/ |
| Journal: | Protein science 32 1 |
| 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 |
Files in This Item:
| File | Description | Size | Format | ||
|---|---|---|---|---|---|
 | a_competition_smfret_assay_to-20240216093225346.pdf | 2.45 MB | Adobe PDF | View/Open |