title: 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.
authors.affiliation: Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China.
authors.forename: Feng-Lan
authors.initials: FL
authors.lastname: Zhao
authors.affiliation: Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China.
authors.forename: Chang
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China.
authors.forename: Chen
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Lab of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China. Electronic address: bcye@ecust.edu.cn.
authors.forename: Bang-Ce
authors.initials: BC
authors.lastname: Ye
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics*; D001426:Bacterial Proteins; D015374:Biosensing Techniques / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000737:chemistry; D008164:Luminescent Proteins; D009249:NADP / Q000032:analysis* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods
chemical_list: D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D009249:NADP; D000429:Alcohol Oxidoreductases; C021934:2-dehydropantoate 2-reductase
keywords: Computational protein redesign; FRET; Genetically encoded biosensor; NADP(+)