Live cell monitoring of glycine betaine by FRET-based genetically encoded nanosensor

Abstract

Glycine betaine (GB) is one of the key compatible solutes that accumulate in the cell at exceedingly high level under the conditions of high salinity. It plays a crucial role in the maintenance of osmolarity of the cell without affecting the physiological processes. Analysis of stress-induced physiological conditions in living cells, therefore, requires real-time monitoring of cellular GB level. Glycine Betaine Optical Sensor (GBOS), a genetically-encoded FRET-based nanosensor developed in this study, allows the real-time monitoring of GB levels inside living cells. This nanosensor has been developed by sandwiching GB binding protein (ProX) between the Förster resonance energy transfer (FRET) pair, the cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). Conformational change in ProX, which was used as sensory domain, reported the change in the level of this compatible solute in in vitro and in vivo conditions. Binding of the GB to the sensory domain fetches close to both the fluorescent moieties that result in the form of increased FRET ratio. So, any change in the concentration of GB is correlated with change in FRET ratio. This sensor also reported the GB cellular dynamics in real-time in Escherichia coli cells after the addition of its precursor, choline. The GBOS was also expressed in yeast and mammalian cells to monitor the intracellular GB. Therefore, the GBOS represents a unique FRET-based nanosensor which allows the non-invasive ratiometric analysis of the GB in living cells.