Visualization of thiamine in living cells using genetically encoded fluorescent nanosensor

Abstract

Thiamine (vitamin B1) plays a fundamental role in energy metabolism. Its deficiency in humans manifest in the form of serious diseases like Beriberi and Wernicke–Korsakoff syndrome. Monitoring of thiamine within living cells in real time is of acute interest in medicine. Fluorescent indicator protein for thiamine (FLIPT) is a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor developed in this study that allows real time monitoring of thiamine levels within living cells. Nanosensor is constructed by sandwiching thiamine binding protein (thiB) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), a FRET pair. Definite conformational change in the sensory domain thiB is depicted as a change in the level of this attuned solute under in vitro and in vivo conditions. This turn out when thiamine binds to sensory domain that brings the donor-acceptor pair in vicinity resulting in increased FRET ratio. Henceforth, any variation in thiamine concentration changes the resultant FRET ratio. The constructed nanosensor was very specific to thiamine and stable to pH within physiological range. The calculated affinity (Kd) of FLIPT is 529 nM. Chimeric protein was expressed in Escherichia coli (E. coli), yeast and mammalian cells to monitor the thiamine levels. Thus, FLIPT is a genetically encoded FRET based nanosensor that allows real time monitoring of thiamine levels within living cells in a non-invasive manner.