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 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.

Impact of Molecular Crowding on Translational Mobility and Conformational Properties of Biological Macromolecules

“…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).…”

“…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.…”

“…In the case investigated here, glucose binding as well as the presence of macromolecular crowders lead to a structural compaction of the sensor construct, which both alter the FRET ratio. The crowder-induced sensor compaction only takes place if macromolecular crowders (PEG and Ficoll with a few up to a few tens of kDa mass 46 ) are used, whereas a compaction in the presence of small crowder agents (e.g., glycerol) was not observed. Importantly, the sensor sensitivity (given by sufficiently large ΔR-values) is at least maintained under crowding conditions.…”

Functional principles of genetically encoded fluorescent biosensors for metabolism and their quantitative use

“…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.…”