Structural Analysis of a Genetically Encoded FRET Biosensor by SAXS and MD Simulations
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 atta...
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MDPI AG
2021-06-01
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| author | Ines Reinartz Mona Sarter Julia Otten Henning Höfig Martina Pohl Alexander Schug Andreas M. Stadler Jörg Fitter |
| author_facet | Ines Reinartz Mona Sarter Julia Otten Henning Höfig Martina Pohl Alexander Schug Andreas M. Stadler Jörg Fitter |
| author_sort | Ines Reinartz |
| collection | DOAJ |
| description | 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., <i>ACS Sensors</i>, 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. |
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| issn | 1424-8220 |
| language | English |
| last_indexed | 2024-03-10T10:21:17Z |
| publishDate | 2021-06-01 |
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| spelling | doaj.art-3d73b935658b4092aef43fd9d3c283512023-11-22T00:25:03ZengMDPI AGSensors1424-82202021-06-012112414410.3390/s21124144Structural Analysis of a Genetically Encoded FRET Biosensor by SAXS and MD SimulationsInes Reinartz0Mona Sarter1Julia Otten2Henning Höfig3Martina Pohl4Alexander Schug5Andreas M. Stadler6Jörg Fitter7Institute for Automation and Applied Informatics, Karlsruhe Institute of Technology, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, GermanyI Physikalisches Institut (IA), AG Biophysik, RWTH Aachen University, 52074 Aachen, GermanyForschungszentrum Jülich, IBG-1, 52426 Jülich, GermanyI Physikalisches Institut (IA), AG Biophysik, RWTH Aachen University, 52074 Aachen, GermanyForschungszentrum Jülich, IBG-1, 52426 Jülich, GermanyJohn von Neumann Institute for Computing, Jülich Supercomputing Centre, Forschungszentrum Jülich, 52428 Jülich, GermanyForschungszentrum Jülich, IBI-8/JCNS-1, 52428 Jülich, GermanyI Physikalisches Institut (IA), AG Biophysik, RWTH Aachen University, 52074 Aachen, GermanyInspired 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., <i>ACS Sensors</i>, 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.https://www.mdpi.com/1424-8220/21/12/4144glucose sensorgreen fluorescence protein (GFP)single-molecule FRETsmall angle X-ray scattering (SAXS)coarse-grained molecular dynamics (MD) |
| spellingShingle | Ines Reinartz Mona Sarter Julia Otten Henning Höfig Martina Pohl Alexander Schug Andreas M. Stadler Jörg Fitter Structural Analysis of a Genetically Encoded FRET Biosensor by SAXS and MD Simulations Sensors glucose sensor green fluorescence protein (GFP) single-molecule FRET small angle X-ray scattering (SAXS) coarse-grained molecular dynamics (MD) |
| title | Structural Analysis of a Genetically Encoded FRET Biosensor by SAXS and MD Simulations |
| title_full | Structural Analysis of a Genetically Encoded FRET Biosensor by SAXS and MD Simulations |
| title_fullStr | Structural Analysis of a Genetically Encoded FRET Biosensor by SAXS and MD Simulations |
| title_full_unstemmed | Structural Analysis of a Genetically Encoded FRET Biosensor by SAXS and MD Simulations |
| title_short | Structural Analysis of a Genetically Encoded FRET Biosensor by SAXS and MD Simulations |
| title_sort | structural analysis of a genetically encoded fret biosensor by saxs and md simulations |
| topic | glucose sensor green fluorescence protein (GFP) single-molecule FRET small angle X-ray scattering (SAXS) coarse-grained molecular dynamics (MD) |
| url | https://www.mdpi.com/1424-8220/21/12/4144 |
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