Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species
| Autor: | Ilona Ritter, Julia Walter, Jörg Fitter, Antonie Schöne, Victoria Steffen, Alexandros Katranidis, Ignacio Vergara Dal Pont, Michele Cerminara, Henning Höfig, Martina Pohl |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Conformational change Monosaccharide Transport Proteins ligand binding fluorescent protein (FP) hinge motion Pharmaceutical Science Biosensing Techniques biosensor Signal Article Analytical Chemistry lcsh:QD241-441 03 medical and health sciences conformational change lcsh:Organic chemistry Drug Discovery Fluorescence Resonance Energy Transfer Physical and Theoretical Chemistry skin and connective tissue diseases single molecule studies Fluorescent Dyes glucose sensor Chemistry Escherichia coli Proteins Organic Chemistry Förster resonance energy transfer (FRET) Ligand (biochemistry) Fluorescence Glucose binding Luminescent Proteins 030104 developmental biology Förster resonance energy transfer Glucose Chemistry (miscellaneous) Periplasmic Binding Proteins ddc:540 Biophysics Molecular Medicine bacteria sense organs Biosensor |
| Zdroj: | Molecules Volume 23 Issue 12 Molecules, Vol 23, Iss 12, p 3105 (2018) Molecules 23(12), 3105-(2018). doi:10.3390/molecules23123105 Molecules : a journal of synthetic chemistry and natural product chemistry 23(12), 3105 (2018). doi:10.3390/molecules23123105 special issue: "Special Issue "Single-Molecule Fluorescence Spectroscopy" / Special Issue Editor: Prof. Jörg Fitter, Guest Editor, Physikalisches Institut (IA), RWTH Aachen, Germany" |
| ISSN: | 1420-3049 |
| Popis: | 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 Fö rster 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. |
| Databáze: | OpenAIRE |
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