GTP is a major regulator of multiple cellular processes, but tools for quantitative evaluation of GTP levels in live cells have not been available. We report the development and characterization of genetically encoded GTP sensors, which we constructed by inserting a circularly permuted yellow fluorescent protein (cpYFP) into a region of the bacterial G protein FeoB that undergoes a GTP-driven conformational change. GTP binding to these sensors results in a ratiometric change in their fluorescence, thereby providing an internally normalized response to changes in GTP levels while minimally perturbing those levels. Mutations introduced into FeoB to alter its affinity for GTP created a series of sensors with a wide dynamic range. Critically, in mammalian cells the sensors showed consistent changes in ratiometric signal upon depletion or restoration of GTP pools. We show that these GTP evaluators (GEVALs) are suitable for detection of spatiotemporal changes in GTP levels in living cells and for high-throughput screening of molecules that modulate GTP levels.

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  1. 1.

    , & Ribosomal translocation: one step closer to the molecular mechanism. ACS Chem. Biol. 4, 93–107 (2009).

  2. 2.

    , & Diversity of G proteins in signal transduction. Science 252, 802–808 (1991).

  3. 3.

    , & G protein pathways. Science 296, 1636–1639 (2002).

  4. 4.

    IFN-inducible GTPases and immunity to intracellular pathogens. Trends Immunol. 25, 601–609 (2004).

  5. 5.

    et al. Analyses of murine GBP homology clusters based on in silico, in vitro and in vivo studies. BMC Genomics 9, 158 (2008).

  6. 6.

    & The guanylate-binding proteins: emerging insights into the biochemical properties and functions of this family of large interferon-induced guanosine triphosphatase. J. Interferon Cytokine Res. 31, 89–97 (2011).

  7. 7.

    Targeting RAS signalling pathways in cancer therapy. Nat. Rev. Cancer 3, 11–22 (2003).

  8. 8.

    et al. Structural basis of GDP release and gating in G protein coupled Fe2+ transport. EMBO J. 28, 2677–2685 (2009).

  9. 9.

    , , , & The membrane protein FeoB contains an intramolecular G protein essential for Fe(II) uptake in bacteria. Proc. Natl. Acad. Sci. USA 99, 16243–16248 (2002).

  10. 10.

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

  11. 11.

    , & A genetically encoded fluorescent reporter of ATP:ADP ratio. Nat. Methods 6, 161–166 (2009).

  12. 12.

    et al. Visualization of ATP levels inside single living cells with fluorescence resonance energy transfer-based genetically encoded indicators. Proc. Natl. Acad. Sci. USA 106, 15651–15656 (2009).

  13. 13.

    Physiological concentrations of purines and pyrimidines. Mol. Cell. Biochem. 140, 1–22 (1994).

  14. 14.

    , , & Fermentation of xylose causes inefficient metabolic state due to carbon/energy starvation and reduced glycolytic flux in recombinant industrial Saccharomyces cerevisiae. PLoS One 8, e69005 (2013).

  15. 15.

    Cellular magnesium homeostasis. Arch. Biochem. Biophys. 512, 1–23 (2011).

  16. 16.

    , , & Design and application of genetically encoded biosensors. Trends Biotechnol. 29, 144–152 (2011).

  17. 17.

    , & Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor. J. Am. Chem. Soc. 133, 10034–10037 (2011).

  18. 18.

    , , , & Guanine ribonucleotide depletion inhibits T cell activation. Mechanism of action of the immunosuppressive drug mizoribine. J. Clin. Invest. 87, 940–948 (1991).

  19. 19.

    et al. A purine nucleotide biosynthesis enzyme guanosine monophosphate reductase is a suppressor of melanoma invasion. Cell Rep. 5, 493–507 (2013).

  20. 20.

    et al. Induction of apoptosis in IL-3-dependent hematopoietic cell lines by guanine nucleotide depletion. Blood 101, 4958–4965 (2003).

  21. 21.

    , , & Effects of guanine nucleotide depletion on cell cycle progression in human T lymphocytes. Blood 91, 2896–2904 (1998).

  22. 22.

    et al. Direct role of nucleotide metabolism in C-MYC-dependent proliferation of melanoma cells. Cell Cycle 7, 2392–2400 (2008).

  23. 23.

    , & Characterization of nucleotide pools as a function of physiological state in Escherichia coli. J. Bacteriol. 190, 718–726 (2008).

  24. 24.

    et al. A LC-MS/MS method for the analysis of intracellular nucleoside triphosphate levels. Pharm. Res. 26, 1504–1515 (2009).

  25. 25.

    et al. Mycophenolic acid-induced GTP depletion also affects ATP and pyrimidine synthesis in mitogen-stimulated primary human T-lymphocytes. Transplantation 69, 890–897 (2000).

  26. 26.

    , , , & In situ simultaneous monitoring of ATP and GTP using a graphene oxide nanosheet-based sensing platform in living cells. Nat. Protoc. 9, 1944–1955 (2014).

  27. 27.

    et al. The lipid kinase PI5P4Kβ is an intracellular GTP sensor for metabolism and tumorigenesis. Mol. Cell 61, 187–198 (2016).

  28. 28.

    et al. Structural reverse genetics study of the PI5P4Kβ-nucleotide complexes reveals the presence of the GTP bioenergetic system in mammalian cells. FEBS J. 283, 3556–3562 (2016).

  29. 29.

    et al. Depletion of deoxyribonucleotide pools is an endogenous source of DNA damage in cells undergoing oncogene-induced senescence. Am. J. Pathol. 182, 142–151 (2013).

  30. 30.

    et al. Microphthalmia-associated transcription factor suppresses invasion by reducing intracellular GTP pools. Oncogene 36, 84–96 (2016).

  31. 31.

    et al. The role of thioredoxin 1 in the mycophenolic acid-induced apoptosis of insulin-producing cells. Cell Death Dis. 4, e721 (2013).

  32. 32.

    , & Cytochrome C and caspase-3/7 are involved in mycophenolic acid-induced apoptosis in genetically engineered PC12 neuronal cells expressing the p53 gene. Iran. J. Pharm. Res. 13, 191–198 (2014).

  33. 33.

    , & A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73 (1999).

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This work was supported by NIH grants CA151128 and GM118933 (R.S.); CA197996 (D.J.S.); CA120244, CA193981 and CA190533 (M.A.N.); Ruth L. Kirschstein National Research Service Award F32CA189622 (A.B.-S.); NIH grant 1F99CA21245501 (H.C.A.); Empire State Development Corporation Krabbe Disease Research Working Capital X561 and Krabbe Disease Research Capital Equipment U446 (M.L.F.); and the Jennifer Linscott Tietgen Foundation (M.A.N.). The pLV-SV4-puro lentiviral vector was obtained from P. Chumakov (Cleveland Clinic).

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Author notes

    • Mitra S Rana
    •  & Archis Bagati

    Present addresses: NICHD, NIH, Bethesda, Maryland, USA (M.S.R.); Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA (A.B.).

    • Anna Bianchi-Smiraglia
    •  & Mitra S Rana

    These authors contributed equally to this work.


  1. Department of Cell Stress Biology, Roswell Park Cancer Institute, Buffalo, New York, USA.

    • Anna Bianchi-Smiraglia
    • , Colleen E Foley
    • , Leslie M Paul
    • , Brittany C Lipchick
    • , Sudha Moparthy
    • , Kalyana Moparthy
    • , Emily E Fink
    • , Archis Bagati
    • , Eugene S Kandel
    •  & Mikhail A Nikiforov
  2. Department of Biochemistry and Center for Biomedical Neuroscience, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.

    • Mitra S Rana
    •  & Rui Sousa
  3. Department of Biochemistry and Neurology, Hunter James Kelly Research Institute, Jacobs School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, New York, USA.

    • Edward Hurley
    •  & Maria Laura Feltri
  4. Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York, USA.

    • Hayley C Affronti
    • , Andrei V Bakin
    •  & Dominic J Smiraglia


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A.B.-S., M.S.R., R.S. and M.A.N. designed the experiments and wrote the manuscript; A.B.-S. and M.S.R. performed most of the experiments and analyzed the data; C.E.F., B.C.L., L.M.P., S.M., K.M., E.E.F. and A.B. performed some of the experiments; H.C.A. performed HPLC analysis; E.H. assisted with the microscopy acquisition and analysis; D.J.S., A.V.B., E.S.K. and M.L.F. supervised part of the study; R.S. and M.A.N. conceived the initial hypothesis and supervised the study. A.B.-S. and M.S.R. contributed equally to this study. All authors discussed the results and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Rui Sousa or Mikhail A Nikiforov.

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