Skip to main content
SpringerLink
Log in

Redox-Sensitive Green Fluorescent Protein: Probes for Dynamic Intracellular Redox Responses. A Review

  • Protocol
  • First Online:
Redox-Mediated Signal Transduction

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 476))

Abstract

The quantification of transient redox events within subcellular compartments, such as those involved in certain signal transduction pathways, requires specific probes with high spatial and temporal resolution. Redox-sensitive variants of the green fluorescent protein (roGFP) have recently been developed that allow the noninvasive monitoring of intracellular thiol-disulfide equilibria. In this chapter, the biophysical properties of these probes are discussed, including recent efforts to enhance their response times. Several recent applications of roGFPs are highlighted, including roGFP expression within Arabidopsis to monitor redox status during root elongation, expression in neurons to measure oxidative stress during ischemia, and targeting of roGFPs to endosomal compartments demonstrating unexpectedly oxidizing potentials within these compartments. Possible future directions for the optimization of roGFPs or new classes of redox-sensitive fluorescent probes are also discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Jacob, C., Knight, I., and Winyard, P. G. (2006) Aspects of the biological redox chemistry of cysteine: from simple redox responses to sophisticated signalling pathways. Biol Chem 387, 1385–1397.

    Article  PubMed  CAS  Google Scholar 

  2. Hwang, C., Sinskey, A. J., and Lodish, H. F. (1992) Oxidized redox state of glutathione in the endoplasmic reticulum. Science 257, 1496–1502.

    Article  PubMed  CAS  Google Scholar 

  3. Schafer, F. Q. and Buettner, G. R. (2001) Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radic Biol Med 30, 1191–1212.

    Article  PubMed  CAS  Google Scholar 

  4. Austin, C. D., Wen, X., Gazzard, L., Nelson, C., Scheller, R. H., and Scales, S. J. (2005) Oxidizing potential of endosomes and lysosomes limits intracellular cleavage of disulfide-based antibody–drug conjugates. Proc Natl Acad Sci USA 102, 17987–17992.

    Article  PubMed  CAS  Google Scholar 

  5. Rhee, S. G., Chang, T. S., Bae, Y. S., Lee, S. R., and Kang, S. W. (2003) Cellular regulation by hydrogen peroxide. J Am Soc Nephrol 14, S211–S215.

    Article  PubMed  CAS  Google Scholar 

  6. Rhee, S. G., Bae, Y. S., Lee, S. R., and Kwon, J. (2000) Hydrogen peroxide: a key messenger that modulates protein phosphorylation through cysteine oxidation. Sci STKE 2000, PE1.

    Article  PubMed  Google Scholar 

  7. Ormo, M., Cubitt, A. B., Kallio, K., Gross, L. A., Tsien, R. Y., and Remington, S. J. (1996) Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395.

    Article  PubMed  CAS  Google Scholar 

  8. Yang, F., Moss, L. G., and Phillips, G. N. J. (1996) The molecular structure of green fluorescent protein. Nat Biotechnol 14, 1246–1251.

    Article  PubMed  CAS  Google Scholar 

  9. Heim, R., Prasher, D. C., and Tsien, R. Y. (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc Natl Acad Sci USA 91, 12501–12504.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  11. Tsien, R. Y. (1998) The green fluorescent protein. Annu Rev Biochem 67, 509–544.

    Article  PubMed  CAS  Google Scholar 

  12. Chattoraj, M., King, B. A., Bublitz, G. U., and Boxer, S. G. (1996) Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer. Proc Natl Acad Sci USA 93, 8362–8367.

    Article  PubMed  CAS  Google Scholar 

  13. Brejc, K., Sixma, T. K., Kitts, P. A., Kain, S. R., Tsien, R. Y., Ormo, M., and Remington, S. J. (1997) Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc Natl Acad Sci USA 94, 2306–2311.

    Article  PubMed  CAS  Google Scholar 

  14. Palm, G. J., Zdanov, A., Gaitanaris, G. A., Stauber, R., Pavlakis, G. N., and Wlodawer, A. (1997) The structural basis for spectral variations in green fluorescent protein. Nat Struct Biol 4, 361–365.

    Article  PubMed  CAS  Google Scholar 

  15. Ward, W. W. and Bokman, S. H. (1982) Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein. Biochemistry 21, 4535–4540.

    Article  PubMed  CAS  Google Scholar 

  16. Ward, W. W., Prentice, H. J., Roth, A. F., Cody, C. W., and Reeves, S. C. (1982) Spectral perturbations of the Aequorea green-fluorescent protein. Photochem Photobiol 35, 803–808.

    Article  CAS  Google Scholar 

  17. Giepmans, B. N., Adams, S. R., Ellisman, M. H., and Tsien, R. Y. (2006) The fluorescent protein toolbox for assessing protein location and function. Science 312, 217–223.

    Article  PubMed  CAS  Google Scholar 

  18. Zhang, J., Campbell, R. E., Ying, A. Y., and Tsien, R. Y. (2002) Creating new fluorescent probes for cell biology. Nat Rev Cell Biol 3, 906–918.

    Article  CAS  Google Scholar 

  19. Grynkiewicz, G., Poenie, M., and Tsien, R.Y. (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260, 3440–3450.

    PubMed  CAS  Google Scholar 

  20. Hanson, G. T., Aggeler, R., Oglesbee, D., Cannon, M., Capaldi, R. A., Tsien, R. Y., and Remington, S. J. (2004) Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem 279, 13044–13053.

    Article  PubMed  CAS  Google Scholar 

  21. Rossignol, R., Gilkerson, R., Aggeler, R., Yamagata, K., Remington, S. J., and Capaldi, R. A. (2004) Energy substrate modulates mitochondrial structure and oxidative capacity in cancer cells. Cancer Res 64, 985–993.

    Article  PubMed  CAS  Google Scholar 

  22. Dooley, C. T., Dore, T. M., Hanson, G. T., Jackson, W. C., Remington, S. J., and Tsien, R. Y. (2004) Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J Biol Chem 279, 22284–22293.

    Article  PubMed  CAS  Google Scholar 

  23. Ostergaard, H., Henriksen, A., Hansen, F. G., and Winther, J. R. (2001) Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein. EMBO J 20, 5853–5862.

    Article  PubMed  CAS  Google Scholar 

  24. Bjornberg, O., Ostergaard, H., and Winther, J. R. (2006) Measuring intracellular redox conditions using GFP-based sensors. Antioxid Redox Signal 8, 354–361.

    Article  PubMed  Google Scholar 

  25. Ostergaard, H., Tachibana, C., Winther, J. R. (2004) Monitoring disulfide bond formation in the eukaryotic cytosol. J Cell Biol 166, 337–345.

    Article  PubMed  CAS  Google Scholar 

  26. Rhee, S. G., Kang, S. W., Jeong, W., Chang, T. S., Yang, K. S., and Woo, H. A. (2005) Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins. Curr Opin Cell Biol 17, 183–189.

    Article  PubMed  CAS  Google Scholar 

  27. Finkel, T. (1998) Oxygen radicals and signaling. Curr Opin Cell Biol 10, 248–253.

    Article  PubMed  CAS  Google Scholar 

  28. Tu, B. P., Ho-Schleyer, S., Travers, K. J., and Weissman, J. S. (2000) Biochemical basis of oxidative protein folding in the endoplasmic reticulum. Science 290, 1571–1574.

    Article  PubMed  CAS  Google Scholar 

  29. Kim, J. R., Yoon, H.W., Kwon, K. S., Lee, S. R., and Rhee, S.G. (2000) Identification of proteins containing cysteine residues that are sensitive to oxidation by hydrogen peroxide at neutral pH. Ana. Biochem 283, 214–221.

    Article  CAS  Google Scholar 

  30. Cannon, M. B., Remington, S. J. (2006) Re-engineering redox-sensitive green fluorescent protein for improved response rate. Protein Sci 15, 45–57.

    Article  PubMed  CAS  Google Scholar 

  31. Rocchia, W., Alexov, E., and Honig, B. (2001) Extending the applicability of the nonlinear Poisson–Boltzmann equation: multiple dielectric constants and multivalent ions. J Phys Chem B 105, 6507–6514.

    Article  CAS  Google Scholar 

  32. Rocchia, W., Sridharan, S., Nicholls, A., Alexov, E., Chiabrera, A., and Honig, B. (2002) Rapid grid-based construction of the molecular surface for both molecules and geometric objects: applications to the finite difference Poisson-Boltzmann method. J Comp Chem 23, 128–137.

    Article  CAS  Google Scholar 

  33. Hansen, R. E., Ostergaard, H., and Winther, J. R. (2005) Increasing the reactivity of an artificial dithiol-disulfide pair through modification of the electrostatic milieu. Biochemistry 44, 5899–5906.

    Article  PubMed  CAS  Google Scholar 

  34. Creighton, T. E. (1975) Interactions between cysteine residues as probes of protein conformation: the disulfide bond between Cys-14 and Cys-38 of the pancreatic trypsin inhibitor. J Mol Biol 96, 767–776.

    Article  PubMed  CAS  Google Scholar 

  35. Nelson, J. W. and Creighton, T. E. (1994) Reactivity and ionization of the active site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo. Biochemistry 33, 5974–5983.

    Article  PubMed  CAS  Google Scholar 

  36. Jiang, K., Schwarzer, C., Lally, E., Zhang, S., Ruzin, S., Machen, T., Remington, S. J., and Feldman, L. (2006) Expression and characterization of a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein) in Arabidopsis. Plant Physiol 141, 397–403.

    Article  PubMed  CAS  Google Scholar 

  37. Joo, J. H., Bae, Y. S., and Lee, J. S. (2001) Role of auxin-induced reactive oxygen species in root gravitropism. Plant Physiol 126, 1055–1060.

    Article  PubMed  CAS  Google Scholar 

  38. Vesce, S., Jekabsons, M. B., Johnson-Caldwell, L. I., and Nicholls, D. G. (2005) Acute glutathione depletion restricts mitochondrial ATP export in cerebellar granule neurons. J Biol Chem 280, 38720–38728.

    Article  PubMed  CAS  Google Scholar 

  39. Calvete, J. A., Newell, D. R., Wright, A. F., and Rose, M. S. (1994) In vitro and in vivo antitumor activity of ZENECA ZD0490, a recombinant ricin A-chain immunotoxin for the treatment of colorectal cancer. Cancer Res 54, 4684–4690.

    PubMed  CAS  Google Scholar 

  40. Liu, C., Tadayoni, B. M., Bourret, L. A., Mattocks, K. M., Derr, S. M., Widdison, W. C., Kedersha, N. L., Ariniello, P. D., Goldmacher, V. S., Lambert, J. M., Blattler, W. A., and Chari, R. V. (1996) Eradication of large colon tumor xenografts by targeted delivery of maytansinoids. Proc Natl Acad Sci USA 93, 8618–8623.

    Article  PubMed  CAS  Google Scholar 

  41. Austin, C. D., De Maziere, A. M., Pisacane, P. I., van Dijk, S. M., Eigenbrot, C., Sliwkowski, M. X., Klumperman, J., and Scheller, R. H. (2004) Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin. Mol Biol Cell 15, 5268–5282.

    Article  PubMed  CAS  Google Scholar 

  42. Aslund, F. and Beckwith, J. (1999) The thioredoxin superfamily: redundancy, specificity and gray-area genomics. J Bacteriol 181, 1375–1379.

    PubMed  CAS  Google Scholar 

  43. DeLano, W. L. (2002) The PyMOL Molecular Graphics System on World Wide Web: http://www.pymol.org.

Download references

Author information

Authors and Affiliations

  1. Department of Biochemistry and Physical Sciences, BYU-Hawaii, Laie, HI, USA

    Mark B. Cannon

  2. Institute of Molecular Biology and Department of Physics, University of Oregon, Eugene, OR

    S. James Remington

Authors
  1. Mark B. Cannon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. James Remington
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Centre for Research in Plant Science, University of the West of England, Bristol, UK

    John T. Hancock Ph.D.

Rights and permissions

Reprints and permissions

Copyright information

© 2008 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Cannon, M.B., James Remington, S. (2008). Redox-Sensitive Green Fluorescent Protein: Probes for Dynamic Intracellular Redox Responses. A Review. In: Hancock, J.T. (eds) Redox-Mediated Signal Transduction. Methods in Molecular Biology™, vol 476. Humana Press. https://doi.org/10.1007/978-1-59745-129-1_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-129-1_4

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-842-3

  • Online ISBN: 978-1-59745-129-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation