A Genetically Encoded Nanosensor For In Vitro And In Vivo Detection Of NADP~+

NADP+ is a key cofactor for electron transfer in the metabolism of all organisms. Numerous dehydrogenases depend on this pyridine nucleotide to exert vital physiological functions in processes such as pentose phosphate pathway and cellular detoxification reactions. In addition, NADP+ also serves as precursor of potent calcium-mobilizing messengers. Mornitoring the dynamics of cytosolic NADP+would contribute to understanding signal transduction and cell metabolism. In the past decades, great efforts have been made to detect NADP+. High-performance liquid chromatography (HPLC) and assay kit of [free NADP+]/[free NADPH] were both applied to monitor NADP+. The analyses require the use of cell extracts and are impossible to study dynamics in intact individual cells.To date, real-time tracking of NADP+ can only be achieved by genetically encoded fluorescent biosensors.Fluorescent biosensors have been developed to measure and visualize a large quantity of small signaling molecules and key metabolites including ATP, a-ketoglutarate, NADH, Ca2+, amino acid, glucose and glutamine.Intracellular metabolites play crucial roles in cellular activities, and real-time detection of them will contribute a lot to understanding cellular metabolism. Inspired by this, herein, we present a novel strategy for tracking of NADP+ in real-time by creating a genetically encoded fluorescent biosensor based on FRET.We designed a novel NADP+ biosensor consisting of an indicator protein ketopantoate reductase (KPR), and two fluorophores, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). In the presence of NADP+, the biosensor showed a concentration-dependent decrease in FRET efficiency. To improve the sensitivity of the sensor, we systematically engineered the lengths of kpr and performed site-specific mutagenesis on it as well as the obtained optimal link according to the result of computational protein redesign. The resulting biosensor (NADPsor) was found to be highly specific to NADP+ with a detection limit of 1?M. This sensor was also employed to monitor the response of E. coli to the precursor of NADP+, nicotinic acid (NA), and the obtained dynamic changes of FRET ratio with time demonstrated the sensor's competence for in vivo analysis of NADP+...