Genetically encoded FRET-based nanosensor for in vivo measurement of leucine.

Besides fundamental role in protein synthesis, leucine has metabolic roles as energy substrates, precursors for synthesis of other amino acids and as a modulator of muscle protein synthesis via the insulin-signaling pathway. Leucine concentration in cell and tissue is temporally dynamic as the metabolism of leucine is regulated through multiple enzymes and transporters. Assessment of cell-type specific activities of transporters and enzymes by physical fractionation is extremely challenging. Therefore, a method of reporting leucine dynamics at the cellular level is highly desirable. Given this, we developed a series of genetically encoded nanosensors for real-time in vivo measurement of leucine at cellular level. A leucine binding periplasmic binding protein (LivK) of Escherichia coli K12 was flanked with CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein) at N-terminus and C-terminus, respectively. The constructed nanosensors allowed in vitro determination of fluorescence resonance energy transfer (FRET) changes in a concentration-dependent manner. These sensors were found to be specific to leucine, and stable to pH-changes within a physiological range. Genetically encoded sensors can be targeted to a specific cell type, and allow dynamic measurement of leucine concentration in bacterial and yeast cells.