Skip to main content
SpringerLink
Log in

Charge transfer in green fluorescent protein

  • Paper
  • Published:
Photochemical & Photobiological Sciences Aims and scope Submit manuscript

Abstract

Charge transfer reactions that contribute to the photoreactions of the wild type green fluorescent protein (GFP) do not occur in the isolated p-hydroxybenzylidene—imidazolidinone chromophore, demonstrating the role of the protein environment. The high quantum efficiency of the fluorescence photocycle that includes excited state proton transfer and the suppression of non-radiative pathways by the protein environment have been correlated with structural dynamics in the chromophore environment. A low quantum efficiency competing phototransformation reaction of GFP is accompanied by both proton and electron transfer, and closely mimics the charge redistribution that is occurring in the fluorescence photocycle. The protein response to this destabilising event has been demonstrated by cryo-trapping of early products in the reaction pathway and is found to be strong even at 100 K, including displacements of chromophore, protein, solvent and a photogenerated CO2 molecule derived from the decarboxylated Glu 222 side chain. We discuss the ramifications of the observation of strong conformational perturbations below the protein dynamical transition at ∼200 K, in view of low temperature work on other light sensitive proteins such as myoglobin and bacteriorhodopsin. The proton and electron transfer in the phototransformation pathway mimics the proton and charge transfer which occurs during the fluorescence cycle, which leads to common structural responses in both photoreactions as shown by ultrafast spectroscopy. We review and discuss literature on light-induced and thermal charge transfer events, focusing on recent findings addressing conformational dynamics and implications for thermodynamic properties.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. R. Y. Tsien, The green fluorescent protein, Annu. Rev. Biochem., 1998, 67, 509–44.

    Article  CAS  PubMed  Google Scholar 

  2. J. Lippincott-Schwartz and G. H. Patterson, Development and use of fluorescent protein markers in living cells, Science, 2003, 300, 87–91.

    Article  CAS  PubMed  Google Scholar 

  3. M. Zimmer, Green fluorescent protein (GFP): applications, structure, and related photophysical behavior, Chem. Rev., 2002, 102, 759–781.

    Article  CAS  PubMed  Google Scholar 

  4. A. Miyawaki, T. Nagai and H. Mizuno, Engineering fluorescent proteins, Adv. Biochem. Eng./Biotechnol., 2005, 95, 1–15.

    CAS  Google Scholar 

  5. F. Yang, L. G. Moss, G. N. Phillips, Jr., The molecular structure of green fluorescent protein, Nat. Biotechnol., 1996, 14, 1246–1251.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. H. Lossau, A. Kummer, R. Heinecke, F. Pöllinger-Dammer, C. Kompa, G. Bieser, T. Jonsson, C. M. Silva, M. M. Yang, D. C. Youvan, M. E. Michel-Beyerle, Time-resolved spectroscopy of wild-type and mutant Green Fluorescent Proteins reveals excited state deprotonation consistent with fluorophore-protein interactions, Chem. Phys., 1996, 213, 1–16.

    Article  CAS  Google Scholar 

  9. J. J. van Thor, G. Y. Georgiev, M. Towrie and J. T. Sage, Ultrafast and low barrier motions in the photoreactions of the green fluorescent protein, J. Biol. Chem., 2005, 280, 33652–33659.

    Article  PubMed  CAS  Google Scholar 

  10. J. J. van Thor, G. Zanetti, K. Ronayne and M. Towrie, Structural events in the photocycle of Green Fluorescent Protein, J. Phys. Chem. B, 2005, 109, 16099–16108.

    Article  PubMed  CAS  Google Scholar 

  11. D. Stoner-Ma, A. A. Jaye, P. Matousek, M. Towrie, S. R. Meech and P. J. Tonge, Observation of Excited-State Proton Transfer in Green Fluorescent Protein using Ultrafast Vibrational Spectroscopy, J. Am. Chem. Soc., 2005, 127, 2864–2865.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  14. J. J. van Thor, T. Gensch, K. J. Hellingwerf and L. N. Johnson, Phototransformation of green fluorescent protein with UV and visible light leads to decarboxylation of glutamate 222, Nat. Struct. Biol., 2002, 9, 37–41.

    Article  PubMed  CAS  Google Scholar 

  15. R. M. Dickson, A. B. Cubitt, R. Y. Tsien and W. E. Moerner, On/off blinking and switching behaviour of single molecules of green fluorescent protein, Nature, 1997, 388, 355–358.

    Article  CAS  PubMed  Google Scholar 

  16. U. Haupts, S. Maiti, P. Schwille and W. W. Webb, Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy, Proc. Natl. Acad. Sci. USA, 1998, 95, 13573–13578.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. P. Schwille, U. Haupts, S. Maiti and W. W. Webb, Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation, Biophys. J., 1999, 77, 2251–2265.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. S. Habuchi, M. Cotlet, T. Gensch, T. Bednarz, S. Haber-Pohlmeier, J. Rozenski, G. Dirix, J. Michiels, J. Vanderleyden, J. Heberle, F. C. De Schryver and J. Hofkens, Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed, J. Am. Chem. Soc., 2005, 127, 8977–8984.

    Article  CAS  PubMed  Google Scholar 

  19. H. Mizuno, T. K. Mal, K. I. Tong, R. Ando, T. Furuta, M. Ikura and A. Miyawaki, Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein, Mol. Cell, 2003, 12, 1051–1058.

    Article  CAS  PubMed  Google Scholar 

  20. K. Nienhaus, G. U. Nienhaus, J. Wiedenmann and H. Nar, Structural basis for photo-induced protein cleavage and green-to-red conversion of fluorescent protein EosFP, Proc. Natl. Acad. Sci. USA, 2005, 102, 9156–9159.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. M. Andresen, M. C. Wahl, A. C. Stiel, F. Grater, L. V. Schafer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmuller, S. W. Hell and S. Jakobs, Structure and mechanism of the reversible photoswitch of a fluorescent protein, Proc. Natl. Acad. Sci. USA, 2005, 102, 13070–13074.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. H. Niwa, S. Inouye, T. Hirano, T. Matsuno, S. Kojima, M. Kubota, M. Ohashi and F. I. Tsuji, Chemical nature of the light emitter of the Aequorea green fluorescent protein, Proc. Natl. Acad. Sci. USA, 1996, 93, 13617–13622.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. M. E. Martin, F. Negri and M. Olivucci, Origin, nature, and fate of the fluorescent state of the green fluorescent protein chromophore at the CASPT2//CASSCF resolution, J. Am. Chem. Soc., 2004, 126, 5452–5464.

    Article  CAS  PubMed  Google Scholar 

  24. P. Altoe, F. Bernardi, M. Garavelli, G. Orlandi and F. Negri, Solvent effects on the vibrational activity and photodynamics of the green fluorescent protein chromophore: a quantum-chemical study, J. Am. Chem. Soc., 2005, 127, 3952–3963.

    Article  CAS  PubMed  Google Scholar 

  25. A. Sinicropi, T. Andruniow, N. Ferre, R. Basosi and M. Olivucci, Properties of the Emitting State of the Green Fluorescent Protein Resolved at the CASPT2//CASSCF/CHARMM Level, J. Am. Chem. Soc., 2005, 127, 11534–11535.

    Article  CAS  PubMed  Google Scholar 

  26. C. Scharnagl, R. Raupp-Kossmann and S. F. Fischer, Molecular basis for pH sensitivity and proton transfer in green fluorescent protein: protonation and conformational substates from electrostatic calculations, Biophys. J., 1999, 77, 1839–1857.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. T. M. Creemers, A. J. Lock, V. V. Subramaniam, T. M. Jovin and S. Volker, Three photoconvertible forms of green fluorescent protein identified by spectral hole-burning, Nat. Struct. Biol., 1999, 6, 557–560.

    Article  CAS  PubMed  Google Scholar 

  28. S. Bonsma, R. Purchase, S. Jezowski, J. Gallus, F. Konz and S. Volker, Green and red fluorescent proteins: photo- and thermally induced dynamics probed by site-selective spectroscopy and hole burning, ChemPhysChem, 2005, 6, 838–849.

    Article  CAS  PubMed  Google Scholar 

  29. J. T. Kennis, D. S. Larsen, I. H. van Stokkum, M. Vengris, J. J. van Thor, R. van Grondelle, Uncovering the hidden ground state of green fluorescent protein, Proc. Natl. Acad. Sci. USA, 2004, 101, 17988–17993.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. G. Jung, J. Wiehler and A. Zumbusch, The photophysics of green fluorescent protein: influence of the key amino acids at positions 65, 203, and 222, Biophys. J., 2005, 88, 1932–1947.

    Article  CAS  PubMed  Google Scholar 

  31. S. Abbruzzetti, E. Grandi, C. Viappiani, S. Bologna, B. Campanini, S. Raboni, S. Bettati and A. Mozzarelli, Kinetics of acid-induced spectral changes in the GFPmut2 chromophore, J. Am. Chem. Soc., 2005, 127, 626–635.

    Article  CAS  PubMed  Google Scholar 

  32. A. M. Saxena, J. B. Udgaonkar and G. Krishnamoorthy, Protein dynamics control proton transfer from bulk solvent to protein interior: a case study with a green fluorescent protein, Protein Sci., 2005, 14, 1787–1799.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. A. B. Cubitt, R. Heim, S. R. Adams, A. E. Boyd, L. A. Gross and R. Y. Tsien, Understanding, improving and using green fluorescent proteins, Trends Biochem. Sci., 1995, 20, 448–455.

    Article  CAS  PubMed  Google Scholar 

  34. M. Chalfie, Y. Tu, G. Euskirchen, W. W. Ward and D. C. Prasher, Green fluorescent protein as a marker for gene expression, Science, 1994, 263, 802–805.

    Article  CAS  PubMed  Google Scholar 

  35. J. J. van Thor, A. J. Pierik, I. Nugteren-Roodzant, A. Xie and K. J. Hellingwerf, Characterization of the photoconversion of green fluorescent protein with FTIR spectroscopy, Biochemistry, 1998, 37, 16915–16921.

    Article  PubMed  Google Scholar 

  36. H. Kolbe, Untersuchungen über die Elektrolyse organischer Verbindungen, Ann. Chem. Pharm., 1849, 69, 257–294.

    Article  Google Scholar 

  37. C. C. Moser, J. M. Keske, K. Warncke, R. S. Farid and P. L. Dutton, Nature of biological electron transfer, Nature, 1992, 355, 796–802.

    Article  CAS  PubMed  Google Scholar 

  38. R. A. Marcus and N. Sutin, Electron transfers in chemistry and biology, Biochim. Biophys. Acta, 1985, 811, 265–322.

    Article  CAS  Google Scholar 

  39. J. J. Hopfield, Electron Transfer between Biological Molecules by Thermally Activated Tunneling, Proc. Natl. Acad. Sci. USA, 1974, 71, 3640–3644.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. J. Jortner, Temperature dependent activation energy for electron transfer between biological molecules, J. Chem. Phys., 1976, 64, 4860–4867.

    Article  CAS  Google Scholar 

  41. C. C. Page, C. C. Moser, X. Chen and P. L. Dutton, Natural engineering principles of electron tunnelling in biological oxidation-reduction, Nature, 1999, 402, 47–52.

    Article  CAS  PubMed  Google Scholar 

  42. A. F. Bell, D. Stoner-Ma, R. M. Wachter and P. J. Tonge, Light-driven decarboxylation of wild-type green fluorescent protein, J. Am. Chem. Soc., 2003, 125, 6919–26.

    Article  CAS  PubMed  Google Scholar 

  43. P. W. Fenimore, H. Frauenfelder, B. H. McMahon and F. G. Parak, Slaving: solvent fluctuations dominate protein dynamics and functions, Proc. Natl. Acad. Sci. USA, 2002, 99, 16047–16051.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. M. A. Lill and V. Helms, Proton shuttle in green fluorescent protein studied by dynamic simulations, Proc. Natl. Acad. Sci. USA, 2002, 99, 2778–2781.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. A. Xie, L. Kelemen, J. Hendriks, B. J. White, K. J. Hellingwerf and W. D. Hoff, Formation of a new buried charge drives a large-amplitude protein quake in photoreceptor activation, Biochemistry, 2001, 40, 1510–1517.

    Article  CAS  PubMed  Google Scholar 

  46. N. M. L. Webber, K. L. and S. R. Meech, Radiationless Relaxation in a Synthetic Analogue of the Green Fluorescent Protein Chromophore, J. Phys. Chem. B, 2001, 105, 1238–1243.

    Article  CAS  Google Scholar 

  47. W. Weber, V. Helms, J. A. McCammon and P. W. Langhoff, Shedding light on the dark and weakly fluorescent states of green fluorescent proteins, Proc. Natl. Acad. Sci. USA, 1999, 96, 6177–6182.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. A. D. Kummer, C. Kompa, H. Lossau, F. Pöllinger-Dammer, M. E. Michel-Beyerle, C. M. Silva, E. J. Bylina, W. J. Coleman, M. M. Yang and D. C. Youvan, Dramatic reduction in fluorescence quantum yield in mutants of Green Fluorescent Protein due to fast internal conversion, Chem. Phys., 1998, 237, 183–193.

    Article  CAS  Google Scholar 

  49. A. Usman, O. F. Mohammed, E. T. Nibbering, J. Dong, K. M. Solntsev and L. M. Tolbert, Excited-state structure determination of the green fluorescent protein chromophore, J. Am. Chem. Soc., 2005, 127, 11214–11215.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Laboratory of Molecular Biophysics, University of Oxford, Rex Richards Building South Parks Road, Oxford, UK, OX1 3QU

    Jasper J. van Thor

  2. Department of Physics and Center for Interdisciplinary Research on Complex Systems, Northeastern University, Boston, Massachusetts, 02115, USA

    J. Timothy Sage

Authors
  1. Jasper J. van Thor
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J. Timothy Sage
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jasper J. van Thor.

Additional information

† This paper was published as part of the special issue on Proton Transfer in Biological Systems.

Rights and permissions

Reprints and permissions

About this article

Cite this article

van Thor, J.J., Sage, J.T. Charge transfer in green fluorescent protein. Photochem Photobiol Sci 5, 597–602 (2006). https://doi.org/10.1039/b516525c

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1039/b516525c

Search

Navigation