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Structural Analysis of a Genetically Encoded FRET Biosensor by SAXS and MD Simulations.


ABSTRACT: 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.

SUBMITTER: Reinartz I 

PROVIDER: S-EPMC8234384 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Structural Analysis of a Genetically Encoded FRET Biosensor by SAXS and MD Simulations.

Reinartz Ines I   Sarter Mona M   Otten Julia J   Höfig Henning H   Pohl Martina M   Schug Alexander A   Stadler Andreas M AM   Fitter Jörg J  

Sensors (Basel, Switzerland) 20210616 12


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 a  ...[more]

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