A genetically encoded biosensor for in vitro and in vivo detection of NADP(.)

Abstract

NADP(+), the oxidized form of nicotinamide adenine dinucleotide phosphate, plays an essential role as a coenzyme in cellular electron transfer reactions. The concentration of NADP(+) in cytoplasm or organelles is dynamic due to its conversion to many important derivatives. To track the NADP(+) concentration in single living cells, we developed a genetically encoded NADP(+) biosensor by inserting a reporter element, ketopantoate reductase (KPR), between the Förster resonance energy transfer (FRET) pair, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). This recombinant sensor showed a NADP(+) concentration-dependent decrease in the fluorescence ratio in vitro assay. In order to optimize this biosensor, we performed peptide-length optimization and site-directed mutagenesis in the binding pocket of KPR guided by predictions from computational protein redesign. This modified biosensor showed a 70% Δratio increase compared to the wild type and was found to be highly specific to NADP(+), with a detection limit of 1 μM. The sensor also reported NADP(+) real-time cellular dynamics in Escherichia coli (E. coli) after the addition of its precursor, nicotinic acid (NA). Altogether, these results demonstrate the feasibility of the biosensor for visualizing NADP(+) both in vitro and in vivo.

Keywords: Computational protein redesign; FRET; Genetically encoded biosensor; NADP(+).