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

Publikationstyp:
Zeitschriftenaufsatz
Metadaten:
Autoren
  • Hannah Maus
  • Gerald Hinze
  • Stefan Josef Hammerschmidt
  • Thomas Basche
  • Tanja Schirmeister
Autoren-URL
https://www.webofscience.com/api/gateway?GWVersion=2&SrcApp=fis-test-1&SrcAuth=WosAPI&KeyUT=WOS:000899676600001&DestLinkType=FullRecord&DestApp=WOS_CPL
DOI
10.1002/pro.4526
eISSN
1469-896X
Externe Identifier
  • Clarivate Analytics Document Solution ID: 7C2VU
  • PubMed Identifier: 36461913
ISSN
0961-8368
Ausgabe der Veröffentlichung
1
Zeitschrift
PROTEIN SCIENCE
Schlüsselwörter
  • allosteric inhibition
  • competition assay
  • conformational change
  • flavivirus
  • NS2B-NS3 protease
  • smFRET
Artikelnummer
ARTN e4526
Datum der Veröffentlichung
2023
Status
Published
Titel
A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease
Sub types
  • Article
Ausgabe der Zeitschrift
32

Datenquelle: Web of Science (Lite)

Andere Metadatenquellen:
Abstract
<jats:title>Abstract</jats:title><jats:p>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 <jats:italic>K</jats:italic><jats:sub><jats:italic>D</jats:italic></jats:sub> value observed is in accordance with the IC<jats:sub>50</jats:sub> determined in the fluorometric assay. Our novel approach appears to be a valuable tool to study conformational transitions of other proteases and enzymes.</jats:p>
Autoren
  • Hannah Maus
  • Gerald Hinze
  • Stefan Josef Hammerschmidt
  • Thomas Basché
  • Tanja Schirmeister
DOI
10.1002/pro.4526
eISSN
1469-896X
ISSN
0961-8368
Ausgabe der Veröffentlichung
1
Zeitschrift
Protein Science
Sprache
en
Online publication date
2022
Datum der Veröffentlichung
2023
Status
Published
Herausgeber
Wiley
Herausgeber URL
http://dx.doi.org/10.1002/pro.4526
Datum der Datenerfassung
2023
Titel
A competition <scp>smFRET</scp> assay to study ligand‐induced conformational changes of the dengue virus protease
Ausgabe der Zeitschrift
32

Datenquelle: Crossref

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 K<sub>D</sub> value observed is in accordance with the IC<sub>50</sub> determined in the fluorometric assay. Our novel approach appears to be a valuable tool to study conformational transitions of other proteases and enzymes.
Addresses
  • Institute for Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University, Mainz, Germany.
Autoren
  • Hannah Maus
  • Gerald Hinze
  • Stefan Josef Hammerschmidt
  • Thomas Basché
  • Tanja Schirmeister
DOI
10.1002/pro.4526
eISSN
1469-896X
Externe Identifier
  • PubMed Identifier: 36461913
  • PubMed Central ID: PMC9793963
Funding acknowledgements
  • University of Marburg: AY037116
Open access
true
ISSN
0961-8368
Ausgabe der Veröffentlichung
1
Zeitschrift
Protein science : a publication of the Protein Society
Schlüsselwörter
  • Dengue Virus
  • Peptide Hydrolases
  • Viral Nonstructural Proteins
  • Antiviral Agents
  • Ligands
  • Fluorescence Resonance Energy Transfer
Sprache
eng
Medium
Print
Open access status
Open Access
Paginierung
e4526
Datum der Veröffentlichung
2023
Status
Published
Publisher licence
CC BY-NC-ND
Datum der Datenerfassung
2022
Titel
A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease.
Sub types
  • Research Support, Non-U.S. Gov't
  • research-article
  • Journal Article
Ausgabe der Zeitschrift
32

Files

https://europepmc.org/articles/PMC9793963?pdf=render

Datenquelle: Europe PubMed Central

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.
Date of acceptance
2022
Autoren
  • Hannah Maus
  • Gerald Hinze
  • Stefan Josef Hammerschmidt
  • Thomas Basché
  • Tanja Schirmeister
Autoren-URL
https://www.ncbi.nlm.nih.gov/pubmed/36461913
DOI
10.1002/pro.4526
eISSN
1469-896X
Externe Identifier
  • PubMed Central ID: PMC9793963
Ausgabe der Veröffentlichung
1
Zeitschrift
Protein Sci
Schlüsselwörter
  • NS2B-NS3 protease
  • allosteric inhibition
  • competition assay
  • conformational change
  • flavivirus
  • smFRET
  • Dengue Virus
  • Peptide Hydrolases
  • Ligands
  • Fluorescence Resonance Energy Transfer
  • Viral Nonstructural Proteins
  • Antiviral Agents
Sprache
eng
Country
United States
Paginierung
e4526
Datum der Veröffentlichung
2023
Status
Published
Datum, an dem der Datensatz öffentlich gemacht wurde
2023
Titel
A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease.
Sub types
  • Journal Article
  • Research Support, Non-U.S. Gov't
Ausgabe der Zeitschrift
32

Datenquelle: PubMed

Author's licence
CC-BY-NC-ND
Autoren
  • Hannah Maus
  • Gerald Hinze
  • Stefan Josef Hammerschmidt
  • Thomas Basché
  • Tanja Schirmeister
Hosting institution
Universitätsbibliothek Mainz
Sammlungen
  • DFG-491381577-H
Resource version
Published version
DOI
10.1002/pro.4526
File(s) embargoed
false
Open access
true
ISSN
1469-896X
Ausgabe der Veröffentlichung
1
Zeitschrift
Protein science
Schlüsselwörter
  • 540 Chemie
  • 540 Chemistry and allied sciences
  • 610 Medizin
  • 610 Medical sciences
Sprache
eng
Open access status
Open Access
Paginierung
4526
Datum der Veröffentlichung
2023
Public URL
https://openscience.ub.uni-mainz.de/handle/20.500.12030/10114
Herausgeber
Wiley
Datum der Datenerfassung
2024
Datum, an dem der Datensatz öffentlich gemacht wurde
2024
Zugang
Public
Titel
A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease
Ausgabe der Zeitschrift
32

Files

a_competition_smfret_assay_to-20240216093225346.pdf

Datenquelle: OPENSCIENCE.UB

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