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Nat Methods. 2016 Apr;13(4):352-8. doi: 10.1038/nmeth.3764. Epub 2016 Feb 15.

Apollo-NADP(+): a spectrally tunable family of genetically encoded sensors for NADP(+).

Author information

1
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.
2
Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada.
3
Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
4
Banting and Best Diabetes Centre, University of Toronto, Toronto, Ontario, Canada.

Abstract

NADPH-dependent antioxidant pathways have a critical role in scavenging hydrogen peroxide (H2O2) produced by oxidative phosphorylation. Inadequate scavenging results in H2O2 accumulation and can cause disease. To measure NADPH/NADP(+) redox states, we explored genetically encoded sensors based on steady-state fluorescence anisotropy due to FRET (fluorescence resonance energy transfer) between homologous fluorescent proteins (homoFRET); we refer to these sensors as Apollo sensors. We created an Apollo sensor for NADP(+) (Apollo-NADP(+)) that exploits NADP(+)-dependent homodimerization of enzymatically inactive glucose-6-phosphate dehydrogenase (G6PD). This sensor is reversible, responsive to glucose-stimulated metabolism and spectrally tunable for compatibility with many other sensors. We used Apollo-NADP(+) to study beta cells responding to oxidative stress and demonstrated that NADPH is significantly depleted before H2O2 accumulation by imaging a Cerulean-tagged version of Apollo-NADP(+) with the H2O2 sensor HyPer.

PMID:
26878383
DOI:
10.1038/nmeth.3764
[Indexed for MEDLINE]

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