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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(+).

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


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.

[Indexed for MEDLINE]

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