Abstract
Inspired by the modular architecture of natural signaling proteins, ligand binding proteins are equipped with two fluorescent proteins (FPs) in order to obtain Förster resonance energy transfer (FRET)-based biosensors. Here, we investigated a glucose sensor where the donor and acceptor FPs were attached to a glucose binding protein using a variety of different linker sequences. For three resulting sensor constructs the corresponding glucose induced conformational changes were measured by small angle X-ray scattering (SAXS) and compared to recently published single molecule FRET results (Höfig et al., ACS Sensors, 2018). For one construct which exhibits a high change in energy transfer and a large change of the radius of gyration upon ligand binding, we performed coarse-grained molecular dynamics simulations for the ligand-free and the ligand-bound state. Our analysis indicates that a carefully designed attachment of the donor FP is crucial for the proper transfer of the glucose induced conformational change of the glucose binding protein into a well pronounced FRET signal change as measured in this sensor construct. Since the other FP (acceptor) does not experience such a glucose induced alteration, it becomes apparent that only one of the FPs needs to have a well-adjusted attachment to the glucose binding protein.
Highlights
In the case of genetically encoded Förster resonance energy transfer (FRET)-based sensors, the sensors are often constructed as fusions of full-length protein chromophores, typically green fluorescent protein variants of different colors, attached via short linkers to the ligand binding protein termini
Since the glucose sensor constructs tend to form aggregates when concentrated to a mg/mL range, a size exclusion chromatography (SEC)-small angle X-ray scattering (SAXS) approach was employed in the present study [20]
Short linkers connecting the MglB with each of the fluorescent proteins (FPs) must be chosen in a way that (i) the distance between both FPs remains close enough to observe FRET changes and (ii) the rotational freedom of one or both FPs should be reduced in order to obtain a better coupling of conformational change to FRET change
Summary
Important representatives of biosensors are molecular structures that link highly specific ligand-binding properties of biological macromolecules, mainly proteins, to a physical observable, which can be quantified by a read-out measurement [1,2,3]. Periplasmic binding proteins (PBPs) offer a large potential, since they provide an allosteric linkage between binding of an effector ligand and a pronounced large-scale hinge-bending movement of two globular domains [4,5]. In the case of genetically encoded Förster resonance energy transfer (FRET)-based sensors, the sensors are often constructed as fusions of full-length protein chromophores, typically green fluorescent protein variants of different colors, attached via short linkers to the ligand binding protein termini. Based on known crystal structures of several PBPs, the distance in the range of 4–5 nm between N and
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