Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A genetically encoded fluorescent reporter of ATP:ADP ratio

Nature Methods volume 6, pages 161–166 (2009)Cite this article

This article has been updated

Abstract

We constructed a fluorescent sensor of adenylate nucleotides by combining a circularly permuted variant of GFP with a bacterial regulatory protein, GlnK1, from Methanococcus jannaschii. The sensor's affinity for Mg-ATP was <100 nM, as seen for other members of the bacterial PII regulator family, a surprisingly high affinity given that normal intracellular ATP concentration is in the millimolar range. ADP bound the same site of the sensor as Mg-ATP, competing with it, but produced a smaller change in fluorescence. At physiological ATP and ADP concentrations, the binding site is saturated, but competition between the two substrates causes the sensor to behave as a nearly ideal reporter of the ATP:ADP concentration ratio. This principle for sensing the ratio of two analytes by competition at a high-affinity site probably underlies the normal functioning of PII regulatory proteins. The engineered sensor, Perceval, can be used to monitor the ATP:ADP ratio during live-cell imaging.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Properties of the GlnK1-cpmVenus QV5 construct.
Figure 2: The QV5 construct reports the ATP:ADP ratio.
Figure 3: Perceval is an improved version of the QV5 construct.
Figure 4: Metabolic inhibition leads to a change in the Perceval signal.
Figure 5: Concurrent Perceval and pH monitoring, with pH correction of the Perceval signal.
Figure 6: Transient glucose removal leads to a reversible change in the ATP:ADP ratio signal.

Similar content being viewed by others

Change history

  • 11 January 2009

    NOTE: In the version of this article initially published online, the labels indicating glucose removal in Figure 6 were misaligned. This error has been corrected for the print, PDF and HTML versions of this article.

References

  1. Ashcroft, F.M. & Gribble, F.M. ATP-sensitive K+ channels and insulin secretion: their role in health and disease. Diabetologia 42, 903–919 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Dennis, P.B. et al. Mammalian TOR: a homeostatic ATP sensor. Science 294, 1102–1105 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Weiss, J.N. & Lamp, S.T. Cardiac ATP-sensitive K+ channels. Evidence for preferential regulation by glycolysis. J. Gen. Physiol. 94, 911–935 (1989).

    Article  CAS  PubMed  Google Scholar 

  4. Hoffman, J.F. ATP compartmentation in human erythrocytes. Curr. Opin. Hematol. 4, 112–115 (1997).

    Article  CAS  PubMed  Google Scholar 

  5. Wilson, T. & Hastings, J.W. Bioluminescence. Annu. Rev. Cell Dev. Biol. 14, 197–230 (1998).

    Article  CAS  PubMed  Google Scholar 

  6. Kennedy, H.J. et al. Glucose generates sub-plasma membrane ATP microdomains in single islet beta-cells. Potential role for strategically located mitochondria. J. Biol. Chem. 274, 13281–13291 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Bell, C.J., Manfredi, G., Griffiths, E.J. & Rutter, G.A. Luciferase expression for ATP imaging: application to cardiac myocytes. Methods Cell Biol. 80, 341–352 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Baird, G.S., Zacharias, D.A. & Tsien, R.Y. Circular permutation and receptor insertion within green fluorescent proteins. Proc. Natl. Acad. Sci. USA 96, 11241–11246 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Nagai, T., Sawano, A., Park, E.S. & Miyawaki, A. Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc. Natl. Acad. Sci. USA 98, 3197–3202 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Belousov, V.V. et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nat. Methods 3, 281–286 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Ninfa, A.J. & Jiang, P. PII signal transduction proteins: sensors of alpha-ketoglutarate that regulate nitrogen metabolism. Curr. Opin. Microbiol. 8, 168–173 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Durand, A. & Merrick, M. In vitro analysis of the Escherichia coli AmtB-GlnK complex reveals a stoichiometric interaction and sensitivity to ATP and 2-oxoglutarate. J. Biol. Chem. 281, 29558–29567 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Yildiz, O., Kalthoff, C., Raunser, S. & Kühlbrandt, W. Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake. EMBO J. 26, 589–599 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ereciñska, M. & Silver, I.A. Ions and energy in mammalian brain. Prog. Neurobiol. 43, 37–71 (1994).

    Article  PubMed  Google Scholar 

  15. Wolfe, D.M., Zhang, Y. & Roberts, G.P. Specificity and regulation of interaction between the PII and AmtB1 proteins in Rhodospirillum rubrum. J. Bacteriol. 189, 6861–6869 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Jiang, P. & Ninfa, A.J. Escherichia coli PII signal transduction protein controlling nitrogen assimilation acts as a sensor of adenylate energy charge in vitro. Biochemistry 46, 12979–12996 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Atkinson, D.E. The energy charge of the adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7, 4030–4034 (1968).

    Article  CAS  PubMed  Google Scholar 

  18. Hardie, D.G., Salt, I.P., Hawley, S.A. & Davies, S.P. AMP-activated protein kinase: an ultrasensitive system for monitoring cellular energy charge. Biochem. J. 338, 717–722 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nilsson, T., Schultz, V., Berggren, P.O., Corkey, B.E. & Tornheim, K. Temporal patterns of changes in ATP/ADP ratio, glucose 6-phosphate and cytoplasmic free Ca2+ in glucose-stimulated pancreatic beta-cells. Biochem. J. 314, 91–94 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. DeVivo, D.C., Leckie, M.P., Ferrendelli, J.S. & McDougal, D.B. Chronic ketosis and cerebral metabolism. Ann. Neurol. 3, 331–337 (1978).

    Article  CAS  PubMed  Google Scholar 

  21. Folbergrová, J., Minamisawa, H., Ekholm, A. & Siesjö, B.K. Phosphorylase alpha and labile metabolites during anoxia: correlation to membrane fluxes of K+ and Ca2+. J. Neurochem. 55, 1690–1696 (1990).

    Article  PubMed  Google Scholar 

  22. Veech, R.L., Lawson, J.W., Cornell, N.W. & Krebs, H.A. Cytosolic phosphorylation potential. J. Biol. Chem. 254, 6538–6547 (1979).

    CAS  PubMed  Google Scholar 

  23. Mörikofer-Zwez, S. & Walter, P. Binding of ADP to rat liver cytosolic proteins and its influence on the ratio of free ATP/free ADP. Biochem. J. 259, 117–124 (1989).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Koretsky, A.P., Brosnan, M.J., Chen, L.H., Chen, J.D. & Dyke, T.V. NMR detection of creatine kinase expressed in liver of transgenic mice: determination of free ADP levels. Proc. Natl. Acad. Sci. USA 87, 3112–3116 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Chiuman, W. & Li, Y. Simple fluorescent sensors engineered with catalytic DNA 'MgZ' based on a non-classic allosteric design. PLoS ONE 2, e1224 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Huizenga, D.E. & Szostak, J.W.A. DNA aptamer that binds adenosine and ATP. Biochemistry 34, 656–665 (1995).

    Article  CAS  PubMed  Google Scholar 

  27. Willemse, M., Janssen, E., de Lange, F., Wieringa, B. & Fransen, J. ATP and FRET–a cautionary note. Nat. Biotechnol. 25, 170–172 (2007).

    Article  CAS  PubMed  Google Scholar 

  28. Kiang, J.G., McKinney, L.C. & Gallin, E.K. Heat induces intracellular acidification in human A-431 cells: role of Na(+)-H+ exchange and metabolism. Am. J. Physiol. 259, C727–C737 (1990).

    Article  CAS  PubMed  Google Scholar 

  29. Brown, S.E., Heming, T.A., Benedict, C.R. & Bidani, A. ATP-sensitive Na(+)-H+ antiport in type II alveolar epithelial cells. Am. J. Physiol. 261, C954–C963 (1991).

    Article  CAS  PubMed  Google Scholar 

  30. Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90 (2002).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank T. Abramson for expert technical assistance with the molecular biology, A. Miyawaki (RIKEN Brain Science Institute) for sending the original plasmid encoding Venus, O. Yildiz and W. Kühlbrandt (Max Planck Institute of Biophysics, Frankfurt am Main) for sending the original plasmid encoding GlnK1, M. Merrick (John Innes Centre) for sending bacterial strains and members of the Yellen lab for their comments and discussion. This work was supported by research grants from the US National Institutes of Health –National Institute of Neurological Disorders and Stroke (NS029693 and NS055031) to G.Y.

Author information

Author notes

  1. Jim Berg

    Present address: Present address: Department of Physiology, University of California, 1550 4th Street, San Francisco, California 94158, USA.,

Authors and Affiliations

  1. Department of Neurobiology, Harvard Medical School, 220 Longwood Ave., Boston, 02115, Massachusetts, USA

    Jim Berg, Yin Pun Hung & Gary Yellen

Authors
  1. Jim Berg
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yin Pun Hung
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gary Yellen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.Y. and J.B. designed the research; G.Y., J.B. and Y.P.H. conducted experiments and wrote the paper.

Corresponding author

Correspondence to Gary Yellen.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Results, Supplementary Methods (PDF 806 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Berg, J., Hung, Y. & Yellen, G. A genetically encoded fluorescent reporter of ATP:ADP ratio. Nat Methods 6, 161–166 (2009). https://doi.org/10.1038/nmeth.1288

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1288

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing