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A genetically encoded fluorescent reporter of ATP:ADP ratio

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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.

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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.

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.

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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.

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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.

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

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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

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