Autoren:	Maus, Hannah Hinze, Gerald Hammerschmidt, Stefan Josef Basché, Thomas Schirmeister, Tanja
Titel:	A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease
Online-Publikationsdatum:	16-Feb-2024
Erscheinungsdatum:	2023
Sprache des Dokuments:	Englisch
Zusammenfassung/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-Sachgruppe:	540 Chemie 540 Chemistry and allied sciences 610 Medizin 610 Medical sciences
Veröffentlichende Institution:	Johannes Gutenberg-Universität Mainz
Organisationseinheit:	FB 09 Chemie, Pharmazie u. Geowissensch.
Veröffentlichungsort:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-10096
Version:	Published version
Publikationstyp:	Zeitschriftenaufsatz
Weitere Angaben zur Dokumentart:	Scientific article
Nutzungsrechte:	CC BY-NC-ND
Informationen zu den Nutzungsrechten:	https://creativecommons.org/licenses/by-nc-nd/4.0/
Zeitschrift:	Protein science 32 1
Seitenzahl oder Artikelnummer:	4526
Verlag:	Wiley
Verlagsort:	Hoboken, NJ
Erscheinungsdatum:	2023
ISSN:	1469-896X
DOI der Originalveröffentlichung:	10.1002/pro.4526
Enthalten in den Sammlungen:	DFG-491381577-H