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

Svea Wilkening; Franz-Josef Schmitt; Marius Horch; Ingo Zebger; Oliver Lenz; Thomas Friedrich

doi:10.1007/s11120-017-0348-0

Photosynthesis Research

September 2017, 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

Authors
Authors and affiliations

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

Original Article

First Online: 06 March 2017

Received: 29 November 2016

Accepted: 01 February 2017

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1 Citations

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

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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)

Appendix

Model for NADH binding and the derivation of K _d

The binding of two NADH molecules to Frex can be described as

$$\text{Frex}+\text{2NADH}\overset{{{K}_{D,1}}}{\longleftrightarrow}\text{FrexN}+\text{NADH}\overset{{{K}_{D,2}}}{\longleftrightarrow}~\text{Frex}{{\text{N}}_{\text{2}}}~$$

For this process, the macroscopic dissociation constant is a product of two microscopic constants, and we shortly denote this product as K _d = K _D,1 × K _D,2. In the Hill fit, we determine the averaged single-step dissociation constant K _D (in the absence of NAD⁺), and the highly cooperative character of NADH binding leads to the valid approximation ${{K}_{d}}\,\approx\,K_{D}^{2}$. FrexN and FrexN₂ refer to Frex with one and two bound NADH molecules, respectively.

Derivation of an equation for inhibition

A model for the inhibition of Frex by NAD⁺ is presented in Fig. 6. If we assume that the fluorescence (F) is only emitted by the species FrexN₂, i.e. Frex bound to two NADH molecules, F relates to the fraction of FrexN₂ by

$$F\sim\frac{\left[ \text{Frex}{{\text{N}}_{2}} \right]}{\left[ \text{Frex} \right]+\left[ \text{Frex}{{\text{N}}_{2}} \right]+\left[ \text{Frex}{{\text{N}}^{+}} \right]}.$$

(9)

Expanding by 1/[Frex] leads to

$$F\sim\frac{\frac{\left[ \text{Frex}{{\text{N}}_{2}} \right]}{\left[ \text{Frex} \right]}}{1+\frac{\left[ \text{Frex}{{\text{N}}_{2}} \right]}{\left[ \text{Frex} \right]}+\frac{\left[ \text{Frex}{{\text{N}}^{+}} \right]}{\left[ \text{Frex} \right]}}.$$

(10)

For the two different reactions, the law of mass action yields

$${{K}_{d}}=\frac{\left[ \text{Frex} \right]{{\left[ \text{N} \right]}^{2}}}{\left[ \text{Frex}{{\text{N}}_{2}} \right]}$$

(11)

and

$${{K}_{I}}=\frac{\left[ \text{Frex} \right]\left[ {{\text{N}}^{+}} \right]}{\left[ \text{Frex}{{\text{N}}^{+}} \right]}.$$

(12)

If we substitute the quotients in Eq. 10 by these expressions, we obtain

$$F\sim\frac{\frac{{{\left[ \text{N} \right]}^{2}}}{{{K}_{d}}}}{1+\frac{{{\left[ \text{N} \right]}^{2}}}{{{K}_{d}}}+\frac{\left[ {{\text{N}}^{+}} \right]}{{{K}_{I}}}}.$$

(13)

Expansion with K _d yields

$$F\sim\frac{{{\left[ \text{N} \right]}^{2}}}{{{K}_{d}}+{{\left[ \text{N} \right]}^{2}}+\frac{\left[ {{\text{N}}^{+}} \right]{{K}_{d}}}{{{K}_{I}}}}$$

(14)

and inclusion of a constant to normalize for the maximum fluorescence (F _max) results in Eq. 15, which equals Eq. 5 that was used to fit the fluorescence data shown in Fig. 7 by its linearized variant F _max/F:

$$F=\frac{{{F}_{\max }}{{\left[ \text{N} \right]}^{2}}}{{{K}_{d}}+{{\left[ \text{N} \right]}^{2}}+\frac{\left[ {{\text{N}}^{+}} \right]{{K}_{d}}}{{{K}_{I}}}}.$$

(15)

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Authors and Affiliations

Svea Wilkening
- 1
Franz-Josef Schmitt
- 1
Email authorView author's OrcID profile
Marius Horch
- 1
Ingo Zebger
- 1
Oliver Lenz
- 1
Thomas Friedrich
- 1

1.Institut für ChemieTechnische Universität BerlinBerlinGermany

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

Abstract

Keywords

Abbreviations

Notes

Acknowledgements

Supplementary material

References

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Cite article

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

Abstract

Keywords

Abbreviations

Notes

Acknowledgements

Supplementary material

References

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Authors and Affiliations

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