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Photosynthesis Research

, Volume 133, Issue 1–3, pp 305–315 | Cite as

Characterization of Frex as an NADH sensor for in vivo applications in the presence of NAD+ and at various pH values

  • Svea Wilkening
  • Franz-Josef SchmittEmail author
  • Marius Horch
  • Ingo Zebger
  • Oliver Lenz
  • Thomas Friedrich
Original Article

Abstract

The fluorescent biosensor Frex, recently introduced as a sensitive tool to quantify the NADH concentration in living cells, was characterized by time-integrated and time-resolved fluorescence spectroscopy regarding its applicability for in vivo measurements. Based on the purified sensor protein, it is shown that the NADH dependence of Frex fluorescence can be described by a Hill function with a concentration of half-maximal sensor response of K D  ≈ 4 µM and a Hill coefficient of n ≈ 2. Increasing concentrations of NADH have moderate effects on the fluorescence lifetime of Frex, which changes by a factor of two from about 500 ps in the absence of NADH to 1 ns under fluorescence-saturating NADH concentrations. Therefore, the observed sevenfold rise of the fluorescence intensity is primarily ascribed to amplitude changes. Notably, the dynamic range of Frex sensitivity towards NADH highly depends on the NAD+ concentration, while the apparent K D for NADH is only slightly affected. We found that NAD+ has a strong inhibitory effect on the fluorescence response of Frex during NADH sensing, with an apparent NAD+ dissociation constant of K I  ≈ 400 µM. This finding was supported by fluorescence lifetime measurements, which showed that the addition of NAD+ hardly affects the fluorescence lifetime, but rather reduces the number of Frex molecules that are able to bind NADH. Furthermore, the fluorescence responses of Frex to NADH and NAD+ depend critically on pH and temperature. Thus, for in vivo applications of Frex, temperature and pH need to be strictly controlled or considered during data acquisition and analysis. If all these constraints are properly met, Frex fluorescence intensity measurements can be employed to estimate the minimum NADH concentration present within the cell at sufficiently low NAD+ concentrations below 100 µM.

Keywords

Fluorescence sensor protein Redox sensing NADH NAD+ Frex Decay-associated spectra Fluorescence lifetime Light-driven biohydrogen production 

Abbreviations

ADP

Adenosine diphosphate

cpFP

Circularly permuted fluorescent protein

cpYFP

Circularly permuted yellow fluorescent protein

DAS

Decay-associated spectra

eGFP

Enhanced green fluorescent protein

DNA

Desoxyribonucleic acid

Frex

Fluorescent Rex

FrexH

Frex of high affinity

IPTG

Isopropyl β-D-1-thiogalactopyranoside

LB

Luria Bertani

NAD

Nicotinamide adenine dinucleotide

NAD+

Oxidized nicotinamide adenine dinucleotide

N+

NAD+

NADH

Reduced nicotinamide adenine dinucleotide

N

NADH

NADP

Nicotinamide adenine dinucleotide phosphate

NADPH

Reduced nicotinamide adenine dinucleotide phosphate

NADP+

Oxidized nicotinamide adenine dinucleotide phosphate

OD

Optical density

PBS

Phosphate-buffered saline

ROS

Reactive oxygen species

rpm

revolutions per minute

TWCSPC

Time- and wavelength-correlated single photon counting

YFP

Yellow fluorescent protein

Notes

Acknowledgements

The authors are grateful to Dr. William Oldham and Prof. Joseph Loscalzo (Harvard Medical School, USA) for providing the Frex(H) expression clones. This work was supported by the German Research Foundation—Cluster of Excellence “Unifying Concepts in Catalysis” (to S.W., M.H., T.F., I.Z., and O.L.).

Supplementary material

11120_2017_348_MOESM1_ESM.docx (211 kb)
Supplementary material 1 (DOCX 212 KB)

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Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  1. 1.Institut für ChemieTechnische Universität BerlinBerlinGermany

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