An Novel Fluorescent NADH Reporter

Nicotinamide adenine dinucleotide (NAD+) and reduced nicotinamide adenine dinuclotide (NADH) have been known as coenzyme found in all living cell. As we all know, NADH play an important role in energy metabolism, signal transduction and disease diagnosis. Thus, the sub-cellular image of cell and tissue distribution of NADH and dynamic change would provide key information for healthcare research. But the traditional measurement was based on intrinsic fluorescence of NADH when excited at 340nm, which was difficult to determine NADH in the presence of NADPH. Thus the lack of effective measurement, which be used to strictly distinguish between NADH and NADPH, limit the in Vivo research related to NADH study. In the present study, we used protein-engineering techniques for the rational design of a fusion protein. Here, we constructed a fluorescent sensor of NADH by combining a variant of GFP with a NADH binding proteinYdiH. To optimize the sensor Perex for high response to NADH and low binding to ohters nucleotides analogue, we designed a site-directed mutagenesis screen on the sensor Perex. Using a high-throughput assay of the fluorescence in response to NADH and analogs, we obtain two sensitive and specific sensor used in different condition. In this theses, we detemined the fluorescence properties of the NAD(H) biosensor Perex under different conditions, including pH, temperature and pyridine nucleotides analogue. The results of cell biology experiment shows that this sensor could express in eukaryotic cells as a tool for prediction of subcellular localization in cells and a indicator for dynamic changes in the physiological level of NADH. Our experiments showed that Perex is a powerful instrument for cell biology, physiology, bioengineering and metabolic engineering etc, used to monitor the spatio-temporal distribution of NADH in vivo, tissue, cell and subcellular organelles.