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Genetically encoded FRET-based biosensors have been developed as a tool to quantify metabolites in living cells. The sensors consist of a central metabolite binding protein and flanking fluorescent GFP proteins affixed by different linker sequences.[1] Fluorescence measurements are sensitive either to distance or orientation changes of GFP domains as response to glucose binding. They were used to determine a change in energy transfer upon glucose binding. SEC(size exclusion chromatography)-SAXS measurements have been performed to investigate, if either large-scale structural change of the GRP positions or relative orientation changes occur as response to glucose binding. Based on the measured SAXS curves we have performed rigid body modelling of the GFP domains. These structures fit with the behaviour of the sensor expected due to fluorescent measurements.
In order to gain a better understanding of the sensor under macromolecular crowding conditions mimicking the cellular state SANS experiments have been performed where the sensor was surrounded by contrast matched partially deuterated PEG 6kDa in different concentrations.
[1] V. Steffen et al, 2016, Sensors, vol. 16, no. 10, p. 1604
