2018
DOI: 10.3390/molecules23123105
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Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species

Abstract: 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 … Show more

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Cited by 2 publications
(4 citation statements)
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References 31 publications
(68 reference statements)
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“…This indicates that besides glucose-induced sensor compaction, there is an independent additional compaction of the whole sensor construct caused by both crowder agents. On the other hand, crowding agents seem not to compact only the glucose binding protein, as we have shown recently . (iii) Ficoll 70 induces a rather uniform compaction of nonliganded and liganded states, with rather similar K D -values and Δ R -values for all crowder concentrations (Figure c).…”
Section: Resultssupporting
confidence: 69%
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“…This indicates that besides glucose-induced sensor compaction, there is an independent additional compaction of the whole sensor construct caused by both crowder agents. On the other hand, crowding agents seem not to compact only the glucose binding protein, as we have shown recently . (iii) Ficoll 70 induces a rather uniform compaction of nonliganded and liganded states, with rather similar K D -values and Δ R -values for all crowder concentrations (Figure c).…”
Section: Resultssupporting
confidence: 69%
“…On the other hand, crowding agents seem not to compact only the glucose binding protein, as we have shown recently. 46 (iii) Ficoll 70 induces a rather uniform compaction of nonliganded and liganded states, with rather similar K D -values and ΔR-values for all crowder concentrations (Figure 5c). The impact of PEG 35 seems to be more elaborate.…”
Section: ■ Results and Discussionmentioning
confidence: 85%
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“…The cellular environment is complex and differs from the controlled in vitro conditions used to characterize sensor performance, which can alter the sensor response (Díaz‐García et al, 2019; Gerkau et al, 2019; Koveal et al, 2022; Looger et al, 2005; Schwarz et al, 2019; Zhao et al, 2020). While the underlying reasons are not fully understood for every sensor, the effect of molecular crowding on FRET sensors and individual fluorescent proteins has been well characterized (Höfig et al, 2018; Morikawa et al, 2016; Schwarz et al, 2019). These effects can be subtle or severe and are evaluated by performing the calibration inside of cells, discussed further in the following section.…”
Section: Introductionmentioning
confidence: 99%