NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems

Pamela E. Molinari, Adriana R. Krapp, Andrea Weiner, Hannes M. Beyer, Arun Kumar Kondadi, Tim Blomeier, Melina López, Pilar Bustos-Sanmamed, Evelyn Tevere, Wilfried Weber, Andreas S. Reichert, Nora B. Calcaterra, Mathias Beller, Nestor Carrillo () and Matias D. Zurbriggen ()
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Pamela E. Molinari: Universidad Nacional de Rosario (UNR)
Adriana R. Krapp: Universidad Nacional de Rosario (UNR)
Andrea Weiner: Universidad Nacional de Rosario (UNR)
Hannes M. Beyer: University of Düsseldorf
Arun Kumar Kondadi: Medical Faculty and University Hospital Düsseldorf, Heinrich-Heine-University Düsseldorf
Tim Blomeier: University of Düsseldorf
Melina López: Universidad Nacional de Rosario (UNR)
Pilar Bustos-Sanmamed: Universidad Nacional de Rosario (UNR)
Evelyn Tevere: Universidad Nacional de Rosario (UNR)
Wilfried Weber: University of Freiburg
Andreas S. Reichert: Medical Faculty and University Hospital Düsseldorf, Heinrich-Heine-University Düsseldorf
Nora B. Calcaterra: Universidad Nacional de Rosario (UNR)
Mathias Beller: University of Düsseldorf
Nestor Carrillo: Universidad Nacional de Rosario (UNR)
Matias D. Zurbriggen: University of Düsseldorf

Nature Communications, 2023, vol. 14, issue 1, 1-18

Abstract: Abstract NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate ENADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxido-reduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research.

Date: 2023
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DOI: 10.1038/s41467-023-38739-4

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