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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.
Highlights
The diversity of biological functions, like ligand binding, conformational changes, or structural adaptability of proteins is used already since many years to engineer biosensors [1]
In order to find the best design of a dye-based Förster resonance energy transfer (FRET) sensor that monitors the glucose-induced conformational changes within the glucose binding protein (MglB), two 3D-structures from the Protein
Since an inter-dye distance RDA close to the Förster radius is most sensitive to conformational changes induced by glucose binding, two surface accessible positions for dye attachment had to be identified which: (i) exhibit an averaged inter-dye distance close to R0 concomitant with (ii) a large difference in RDA between the ligand-free and the ligand-bound structures
Summary
The diversity of biological functions, like ligand binding, conformational changes, or structural adaptability of proteins is used already since many years to engineer biosensors [1]. PBPs consist of two domains connected by a hinge region which includes a ligand binding site located at the interface of the two flanking domains. Depending on the ligand binding status the whole structure can adopt two different conformations: a ligand-free open form and a ligand-bound closed form, which interconvert through a bending and a swiveling twist motion about the hinge [2,3,4,5]. Ligand binding was reported as changes in fluorescence intensity, for example by local quenching effects.
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