Journal of Biological Chemistry
Volume 291, Issue 5, 29 January 2016, Pages 2260-2269
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Protein Structure and Folding
Visualization of NO3/NO2 Dynamics in Living Cells by Fluorescence Resonance Energy Transfer (FRET) Imaging Employing a Rhizobial Two-component Regulatory System*

https://doi.org/10.1074/jbc.M115.687632Get rights and content
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Abstract

Nitrate (NO3) and nitrite (NO2) are the physiological sources of nitric oxide (NO), a key biological messenger molecule. NO3/NO2 exerts a beneficial impact on NO homeostasis and its related cardiovascular functions. To visualize the physiological dynamics of NO3/NO2 for assessing the precise roles of these anions, we developed a genetically encoded intermolecular fluorescence resonance energy transfer (FRET)-based indicator, named sNOOOpy (sensor for NO3/NO2 in physiology), by employing NO3/NO2-induced dissociation of NasST involved in the denitrification system of rhizobia. The in vitro use of sNOOOpy shows high specificity for NO3 and NO2, and its FRET signal is changed in response to NO3/NO2 in the micromolar range. Furthermore, both an increase and decrease in cellular NO3 concentration can be detected. sNOOOpy is very simple and potentially applicable to a wide variety of living cells and is expected to provide insights into NO3/NO2 dynamics in various organisms, including plants and animals.

bacterial signal transduction
biosensor
fluorescence resonance energy transfer (FRET)
in vivo imaging
nitrogen metabolism
protein-protein interaction
NO3/NO2 in physiology

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*

The authors declare that they have no conflicts of interest with the contents of this article.

This article was selected as a Paper of the Week.

This article contains supplemental Movies S1 and S2.

1

Supported by Grants-in-aid for Young Scientist (B) 24780092 and 26850042 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

2

Both authors contributed equally to this work.

3

Supported by BRAIN and Grant-in-aid for Scientific Research (A) 26252065 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.