Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species
Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energ...
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| Format: | Article |
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MDPI AG
2018-11-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/23/12/3105 |
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| author | Henning Höfig Michele Cerminara Ilona Ritter Antonie Schöne Martina Pohl Victoria Steffen Julia Walter Ignacio Vergara Dal Pont Alexandros Katranidis Jörg Fitter |
| author_facet | Henning Höfig Michele Cerminara Ilona Ritter Antonie Schöne Martina Pohl Victoria Steffen Julia Walter Ignacio Vergara Dal Pont Alexandros Katranidis Jörg Fitter |
| author_sort | Henning Höfig |
| collection | DOAJ |
| description | Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor. |
| first_indexed | 2024-12-12T20:30:12Z |
| format | Article |
| id | doaj.art-8c5fba5378884af789a6d7139d327d87 |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-12-12T20:30:12Z |
| publishDate | 2018-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-8c5fba5378884af789a6d7139d327d872022-12-22T00:13:03ZengMDPI AGMolecules1420-30492018-11-012312310510.3390/molecules23123105molecules23123105Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled SpeciesHenning Höfig0Michele Cerminara1Ilona Ritter2Antonie Schöne3Martina Pohl4Victoria Steffen5Julia Walter6Ignacio Vergara Dal Pont7Alexandros Katranidis8Jörg Fitter9Forschungszentrum Jülich, ICS-5, 52425 Jülich, GermanyForschungszentrum Jülich, ICS-5, 52425 Jülich, GermanyForschungszentrum Jülich, ICS-5, 52425 Jülich, GermanyForschungszentrum Jülich, ICS-5, 52425 Jülich, GermanyForschungszentrum Jülich, IBG-1, 52425 Jülich, GermanyForschungszentrum Jülich, IBG-1, 52425 Jülich, GermanyRWTH Aachen University, I. Physikalisches Institut (IA), 52056 Aachen, GermanyForschungszentrum Jülich, ICS-5, 52425 Jülich, GermanyForschungszentrum Jülich, ICS-5, 52425 Jülich, GermanyForschungszentrum Jülich, ICS-5, 52425 Jülich, GermanyBacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor.https://www.mdpi.com/1420-3049/23/12/3105Förster resonance energy transfer (FRET)single molecule studiesbiosensorfluorescent protein (FP)conformational changehinge motionligand bindingglucose sensor |
| spellingShingle | Henning Höfig Michele Cerminara Ilona Ritter Antonie Schöne Martina Pohl Victoria Steffen Julia Walter Ignacio Vergara Dal Pont Alexandros Katranidis Jörg Fitter Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species Molecules Förster resonance energy transfer (FRET) single molecule studies biosensor fluorescent protein (FP) conformational change hinge motion ligand binding glucose sensor |
| title | Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species |
| title_full | Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species |
| title_fullStr | Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species |
| title_full_unstemmed | Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species |
| title_short | Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species |
| title_sort | single molecule studies on a fret biosensor lessons from a comparison of fluorescent protein equipped versus dye labeled species |
| topic | Förster resonance energy transfer (FRET) single molecule studies biosensor fluorescent protein (FP) conformational change hinge motion ligand binding glucose sensor |
| url | https://www.mdpi.com/1420-3049/23/12/3105 |
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