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Structural analysis of a genetically encoded fret biosensor by SAXS and MD simulations

Reinartz, Ines 1; Sarter, Mona; Otten, Julia; Höfig, Henning; Pohl, Martina; Schug, Alexander; Stadler, Andreas M.; Fitter, Jörg
1 Karlsruher Institut für Technologie (KIT)


Inspired by the modular architecture of natural signaling proteins, ligand binding proteins are equipped with two fluorescent proteins (FPs) in order to obtain Förster resonance energy transfer (FRET)-based biosensors. Here, we investigated a glucose sensor where the donor and acceptor FPs were attached to a glucose binding protein using a variety of different linker sequences. For three resulting sensor constructs the corresponding glucose induced conformational changes were measured by small angle X-ray scattering (SAXS) and compared to recently published single molecule FRET results (Höfig et al., ACS Sensors, 2018). For one construct which exhibits a high change in energy transfer and a large change of the radius of gyration upon ligand binding, we performed coarse-grained molecular dynamics simulations for the ligand-free and the ligand-bound state. Our analysis indicates that a carefully designed attachment of the donor FP is crucial for the proper transfer of the glucose induced conformational change of the glucose binding protein into a well pronounced FRET signal change as measured in this sensor construct. Since the other FP (acceptor) does not experience such a glucose induced alteration, it becomes apparent that only one of the FPs needs to have a well-adjusted attachment to the glucose binding protein.

Verlagsausgabe §
DOI: 10.5445/IR/1000134272
Veröffentlicht am 26.06.2021
DOI: 10.3390/s21124144
Zitationen: 6
Zitationen: 7
Cover der Publikation
Zugehörige Institution(en) am KIT Institut für Automation und angewandte Informatik (IAI)
Publikationstyp Zeitschriftenaufsatz
Publikationsjahr 2021
Sprache Englisch
Identifikator ISSN: 1424-8220
KITopen-ID: 1000134272
HGF-Programm 47.14.02 (POF IV, LK 01) Information Storage and Processing in the Cell Nucleus
Erschienen in Sensors
Verlag MDPI
Band 21
Heft 12
Seiten Art.Nr. 4144
Vorab online veröffentlicht am 16.06.2021
Schlagwörter glucose sensor; green fluorescence protein (GFP); single-molecule FRET; small angle X-ray scattering (SAXS); coarse-grained molecular dynamics (MD)
Nachgewiesen in Dimensions
Web of Science
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