Skip to main content
SpringerLink
Log in

Diffusion pathways of oxygen species in the phototoxic fluorescent protein Killer Red

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

Abstract

The fluorescent protein KillerRed generates reactive oxygen species through the CALI effect. This property paves the way for the design of genetically encoded photosensitizers for use in cell killing and cancer photodynamic therapy. In this article, we have investigated the diffusion pathways of di-oxygen and the superoxide radical in KillerRed, using molecular dynamics simulations. Our results suggest that, by comparison to the Ser-65-Thr mutant of GFP, diffusion of molecular oxygen (and singlet oxygen) is greatly facilitated in KillerRed, mostly due to the presence of a unique water-filled channel. In contrast, due to their negative charge, superoxide radical ions putatively produced inside the chromophore pocket are unable to escape the protein. These results are consistent with the hypothesis that superoxide generation, if it occurs, proceeds via light-induced photoreduction of the chromophore followed by long-range electron transfer, a mechanism in which the long hydrogen bond network through the channel could play a key role. Alternatively, the facilitated diffusion of di-oxygen through the channel suggests that singlet di-oxygen could be the principal cause of specific CALI of fused proteins. The entry of di-oxygen through the channel probably also accounts for the high susceptibility of KillerRed to photobleaching.

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

Abbreviations

GFP:

Green Fluorescent Protein

EGFP:

Enhanced Green Fluorescent Protein

LES:

Locally Enhanced Sampling

CALI:

Chromophore-Assisted Light Inactivation

PDB:

Protein Data Bank

ROS:

Reactive Oxygen Species

RMSD:

Root Mean Square Deviation

References

  1. M. E. Bulina, D. M. Chudakov, O. V. Britanova, Y. G. Yanushevich, D. B. Staroverov, T. V. Chepurnykh, E. M. Merzlyak, M. A. Shkrob, S. Lukyanov, K. A. Lukyanov, Nat. Biotechnol., 2006, 24, 95–99.

    Article  CAS  Google Scholar 

  2. K. Jacobson, Z. Rajfur, E. Vitriol, K. Hahn, Trends Cell Biol., 2008, 18, 443–450.

    Article  CAS  Google Scholar 

  3. B. A. Griffin, S. R. Adams, R. Y. Tsien, Science, 1998, 281, 269–272.

    Article  CAS  Google Scholar 

  4. S. R. Adams, R. E. Campbell, L. A. Gross, B. R. Martin, G. K. Walkup, Y. Yao, J. Llopis, R. Y. Tsien, J. Am. Chem. Soc., 2002, 124, 6063–6076.

    Article  CAS  Google Scholar 

  5. M. E. Bulina, K. A. Lukyanov, O. V. Britanova, D. Onichtchouk, S. Lukyanov, D. M. Chudakov, Nat. Protoc., 2006, 1, 947–953.

    Article  CAS  Google Scholar 

  6. E. O. Serebrovskaya, E. F. Edelweiss, O. A. Stremovskiy, K. A. Lukyanov, D. M. Chudakov, S. M. Deyev, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 9221–9225.

    Article  CAS  Google Scholar 

  7. E. Horstkotte, T. Schroder, J. Niewohner, E. Thiel, D. G. Jay, S. W. Henning, Photochem. Photobiol., 2005, 81, 358–366.

    Article  CAS  Google Scholar 

  8. A. Jimenez-Banzo, S. Nonell, J. Hofkens, C. Flors, Biophys. J., 2008, 94, 168–172.

    Article  CAS  Google Scholar 

  9. D. Yarbrough, R. M. Wachter, K. Kallio, M. V. Matz, S. J. Remington, Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 462–467.

    Article  CAS  Google Scholar 

  10. S. Pletnev, N. G. Gurskaya, N. V. Pletneva, K. A. Lukyanov, D. M. Chudakov, V. I. Martynov, V. O. Popov, M. V. Kovalchuk, A. Wlodawer, Z. Dauter, V. Pletnev, J. Biol. Chem., 2009, 284, 32028–32039.

    Article  CAS  Google Scholar 

  11. V. Adam, P. Carpentier, S. Violot, M. Lelimousin, C. Darnault, G. U. Nienhaus, D. Bourgeois, J. Am. Chem. Soc., 2009, 131, 18063–18065.

    Article  CAS  Google Scholar 

  12. F. V. Subach, V. N. Malashkevich, W. D. Zencheck, H. Xiao, G. S. Filonov, S. C. Almo, V. V. Verkhusha, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 21097–21102.

    Article  CAS  Google Scholar 

  13. A. M. Bogdanov, A. S. Mishin, I. V. Yampolsky, V. V. Belousov, D. M. Chudakov, F. V. Subach, V. V. Verkhusha, S. Lukyanov, K. A. Lukyanov, Nat. Chem. Biol., 2009, 5, 459–461.

    Article  CAS  Google Scholar 

  14. P. Carpentier, S. Violot, L. Blanchoin, D. Bourgeois, FEBS Lett., 2009, 583, 2839–2842.

    Article  CAS  Google Scholar 

  15. F. V. Subach, G. H. Patterson, M. Renz, J. Lippincott-Schwartz, V. V. Verkhusha, J. Am. Chem. Soc., 2010, 132, 6481–6491.

    Article  CAS  Google Scholar 

  16. R. Heim, D. C. Prasher, R. Y. Tsien, Proc. Natl. Acad. Sci. U. S. A., 1994, 91, 12501–12504.

    Article  CAS  Google Scholar 

  17. A. G. Evdokimov, M. E. Pokross, N. S. Egorov, A. G. Zaraisky, I. V. Yampolsky, E. M. Merzlyak, A. N. Shkoporov, I. Sander, K. A. Lukyanov, D. M. Chudakov, EMBO Rep., 2006, 7, 1006–1012.

    Article  CAS  Google Scholar 

  18. K. Nienhaus, F. Renzi, B. Vallone, J. Wiedenmann, G. U. Nienhaus, Biochemistry, 2006, 45, 12942–12953.

    Article  CAS  Google Scholar 

  19. R. M. Wachter, M. A. Elsliger, K. Kallio, G. T. Hanson, S. J. Remington, Structure, 1998, 6, 1267–1277.

    Article  CAS  Google Scholar 

  20. R. Elber, M. Karplus, J. Am. Chem. Soc., 1990, 112, 9161–9175.

    Article  CAS  Google Scholar 

  21. R. Heim, A. B. Cubitt, R. Y. Tsien, Nature, 1995, 373, 663–664.

    Article  CAS  Google Scholar 

  22. J. C. Phillips, R. Braun, W. Wang, J. Gumbart, E. Tajkhorshid, E. Villa, C. Chipot, R. D. Skeel, L. Kale, K. Schulten, J. Comput. Chem., 2005, 26, 1781–1802.

    Article  CAS  Google Scholar 

  23. M. Brunori, F. Cutruzzola, C. Savino, C. Travaglini-Allocatelli, B. Vallone, Q. H. Gibson, Biophys. J., 1999, 76, 1259–1269.

    Article  CAS  Google Scholar 

  24. J. Cohen, A. Arkhipov, R. Braun, K. Schulten, Biophys. J., 2006, 91, 1844–1857.

    Article  CAS  Google Scholar 

  25. P. Amara, P. Andreoletti, H. M. Jouve, M. J. Field, Protein Sci., 2001, 10, 1927–1935.

    Article  CAS  Google Scholar 

  26. A. D. Mackerell Jr., D. Bashford, M. Bellott, R. L. Dunbrack Jr., J. D. Evanseck, M. J. Field, S. Fischer, J. Gao, H. Guo, S. Ha, J. Phys. Chem. B, 1998, 102, 3586–3616.

    Article  CAS  Google Scholar 

  27. N. Reuter, H. Lin, W. Thiel, J. Phys. Chem. B, 2002, 106, 6310–6321.

    Article  CAS  Google Scholar 

  28. W. Humphrey, A. Dalke, K. Schulten, J. Mol. Graphics, 1996, 14, 33–38.

    Article  CAS  Google Scholar 

  29. M. Lelimousin, M. Noirclerc-Savoye, C. Lazareno-Saez, B. Paetzold, S. Le Vot, R. Chazal, P. Macheboeuf, M. J. Field, D. Bourgeois, A. Royant, Biochemistry, 2009, 48, 10038–10046.

    Article  CAS  Google Scholar 

  30. G. J. Kleywegt, T. A. Jones, Acta Crystallogr., Sect. D: Biol. Crystallogr., 1994, 50, 178–185.

    Article  CAS  Google Scholar 

  31. Collaborative Computational Project, Number 4, The CCP4 suite: programs for protein crystallography, Acta Crystallogr., Sect. D: Biol. Crystallogr., 1994, 50, 760–763.

    Article  Google Scholar 

  32. R. Y. Tsien, Annu. Rev. Biochem., 1998, 67, 509–544.

    Article  CAS  Google Scholar 

  33. M. Gardès-Albert, D. Bonnefont–Rousselot, Z. Abedinzadeh, D. Jore, Actual. Chim., 2003, 269–270, 91–96.

    Google Scholar 

  34. S. Luo, R. L. Levine, FASEB J., 2008, 23, 464–472.

    Article  Google Scholar 

  35. F. Wilkinson, W. P. Helman, A. B. Ross, J. Phys. Chem. Ref. Data, 1995, 24, 663–680.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. IBS, Institut de Biologie Structurale Jean-Pierre Ebel, CEA, CNRS, Université Joseph Fourier, 41 rue Jules Horowitz, F-38027, Grenoble, France

    Arijit Roy, Philippe Carpentier, Dominique Bourgeois & Martin Field

Authors
  1. Arijit Roy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Philippe Carpentier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dominique Bourgeois
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Martin Field
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Dominique Bourgeois or Martin Field.

Additional information

This article is published as part of a themed issue on photofunctional proteins: from understanding to engineering.

‡ Electronic supplementary information (ESI) available: Distance, energy and RMSD analyses of the molecular dynamics simulations. See DOI: 10.1039/c0pp00141d

Rights and permissions

Reprints and permissions

About this article

Cite this article

Roy, A., Carpentier, P., Bourgeois, D. et al. Diffusion pathways of oxygen species in the phototoxic fluorescent protein Killer Red. Photochem Photobiol Sci 9, 1342–1350 (2010). https://doi.org/10.1039/c0pp00141d

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1039/c0pp00141d

Search

Navigation