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The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins

Nature Biotechnology volume 22, pages 289–296 (2004)Cite this article

Abstract

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its fluorescent homologs from Anthozoa corals have become invaluable tools for in vivo imaging of cells and tissues. Despite spectral and chromophore diversity, about 100 cloned members of the GFP-like protein family possess common structural, biochemical and photophysical features. Anthozoa GFP-like proteins are available in colors and properties unlike those of A. victoria GFP variants and thus provide powerful new fluorophores for molecular labeling and intracellular detection. Although Anthozoa GFP-like proteins provide some advantages over GFP, they also have certain drawbacks, such as obligate oligomerization and slow or incomplete fluorescence maturation. In the past few years, effective approaches for eliminating some of these limitations have been described. In addition, several Anthozoa GFP-like proteins have been developed into novel imaging agents, such as monomeric red and dimeric far-red fluorescent proteins, fluorescent timers and photoconvertible fluorescent labels. Future studies on the structure of this diverse set of proteins will further enhance their use in animal tissues and as intracellular biosensors.

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Figure 1: Spectral diversity of Anthozoa GFP-like proteins.
Figure 2: Variety of chromophores within GFP-like proteins.
Figure 3: Monitoring temporal and spatial patterns of target events using fluorescent timer
Figure 4: Overall DsRed tetramer structure.
Figure 5: Ways to overcome FP tetramerization.
Figure 6: Tracking intracellular organelles tagged with kindling fluorescent protein.

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References

  1. Prasher, D.C. et al. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229–233 (1992).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  3. Matz, M.V. et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973 (1999).

    Article  CAS  PubMed  Google Scholar 

  4. Fradkov, A.F. et al. Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence. FEBS Lett. 479, 127–130 (2000).

    Article  CAS  PubMed  Google Scholar 

  5. Wiedenmann, J. et al. Cracks in the beta-can: fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria). Proc. Natl. Acad. Sci. USA 97, 14091–14096 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Labas, Y.A. et al. Diversity and evolution of the green fluorescent protein family. Proc. Natl. Acad. Sci. USA 99, 4256–4261 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Lukyanov, K.A. et al. Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J. Biol. Chem. 275, 25879–25882 (2000).

    Article  CAS  PubMed  Google Scholar 

  8. Gurskaya, N.G. et al. GFP-like chromoproteins as a source of far-red fluorescent proteins. FEBS Lett. 507, 16–20 (2001).

    Article  CAS  PubMed  Google Scholar 

  9. Schlichter, D. et al. Light harvesting by wavelength transformation in symbiotic coral of the Red Sea twilight zone. Mar. Biol. 91, 403–407 (1986).

    Article  Google Scholar 

  10. Salih, A. et al. Fluorescent pigments in corals are photoprotective. Nature 408, 850–853 (2000).

    Article  CAS  PubMed  Google Scholar 

  11. Wilson. T. & Hastings, J.W. Bioluminescence. Annu. Rev. Cell Dev. Biol. 14, 197–230 (1998).

    Article  CAS  PubMed  Google Scholar 

  12. Heim, R. et al. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 91, 12501–12504 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Chalfie, M. et al. Green fluorescent protein as a marker for gene expression. Science 263, 802–805 (1994).

    Article  CAS  PubMed  Google Scholar 

  14. Cubitt, A.B. et al. Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20, 448–455 (1995).

    Article  CAS  PubMed  Google Scholar 

  15. Fukuda, H. et al. Folding of green fluorescent protein and the cycle3 mutant. Biochemistry 39, 12025–12032 (2000).

    Article  CAS  PubMed  Google Scholar 

  16. Zhang, J. et al. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell. Biol. 3, 906–918 (2002).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  19. Ando, R. et al. An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 12651–12656 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Dove, S.G. et al. Major color patterns of reef-building corals are due to a family of GFP-like proteins. Coral Reefs 19, 197–204 (2001).

    Article  Google Scholar 

  21. Remington, S.J. Structural basis for understanding spectral variations in green fluorescent protein. Methods Enzymol. 305, 196–211 (2000).

    Article  CAS  PubMed  Google Scholar 

  22. Wall, M.A. et al. The structural basis for red fluorescence in the tetrameric GFP homolog DsRed. Nat. Struct. Biol. 7, 1133–1138 (2000).

    Article  CAS  PubMed  Google Scholar 

  23. Yarbrough, D. et al. Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-A resolution. Proc. Natl. Acad. Sci. USA 98, 462–467 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Gross, L.A. et al. The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11990–11995 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Prescott, M. et al. The 2.2 Å crystal structure of a pocilloporin pigment reveals a nonplanar chromophore conformation. Structure 11, 275–284 (2003).

    Article  CAS  PubMed  Google Scholar 

  26. Petersen, J. et al. The 2.0 A crystal structure of eqFP611, a far-red fluorescent protein from the sea anemone Entacmaea quadricolor. J. Biol. Chem. 278, 44626–44631 (2003).

    Article  CAS  PubMed  Google Scholar 

  27. Chudakov, D.M. et al. Chromophore environment provides clue to “kindling fluorescent protein” riddle. J. Biol. Chem. 278, 7215–7219 (2003).

    Article  CAS  PubMed  Google Scholar 

  28. Mizuno, H. et al. Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. Molecular Cell 12, 1051–1058 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Baird, G.S. et al. Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97, 11984–11989 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Terskikh, A. et al. “Fluorescent timer”: protein that changes color with time. Science 290, 1585–1588 (2000).

    Article  CAS  PubMed  Google Scholar 

  31. Wiehler, J. et al. Mutants of Discosoma red fluorescent protein with a GFP-like chromophore. FEBS Lett. 487, 384–389 (2001).

    Article  CAS  PubMed  Google Scholar 

  32. Mizuno, H. et al. Red fluorescent protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer. Biochemistry 40, 2502–2510 (2001).

    Article  CAS  PubMed  Google Scholar 

  33. Terskikh, A.V. et al. Analysis of DsRed mutants. Space around the fluorophore accelerates fluorescence development. J. Biol. Chem. 277, 7633–7636 (2002).

    Article  CAS  PubMed  Google Scholar 

  34. Gurskaya, N.G. et al. Color transitions in coral's fluorescent proteins by site-directed mutagenesis. BMC Biochem. 2, 6 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Bulina, M.E. et al. Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis. BMC Biochem. 3, 7 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Duncan, R.R. et al. Functional and spatial segregation of secretory vesicle pools according to vesicle age. Nature 422, 176–180 (2003).

    Article  CAS  PubMed  Google Scholar 

  37. Bertera, S. et al. Body window-enabled in vivo multicolor imaging of transplanted mouse islets expressing an insulin-Timer fusion protein. Biotechniques 35, 718–722 (2003).

    Article  CAS  PubMed  Google Scholar 

  38. Wiegand, U.K. et al. Red, yellow, green go! A novel tool for microscopic segregation of secretory vesicle pools according to their age. Biochem. Soc. Trans. 31, 851–856 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Vrzheshch, P.V. et al. Denaturation and partial renaturation of a tightly tetramerized DsRed protein under mildly acidic conditions. FEBS Lett. 487, 203–208 (2000).

    Article  CAS  PubMed  Google Scholar 

  40. Heikai, A.A. et al. Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine). Proc. Natl. Acad. Sci. USA 97, 11996–12001 (2000).

    Article  Google Scholar 

  41. Verkhusha, V.V. et al. Kinetic analysis of maturation and denaturation of DsRed, a coral-derived red fluorescent protein. Biochemistry (Mosc.) 66, 1342–1351 (2001).

    Article  CAS  Google Scholar 

  42. Wiedenmann, J. et al. A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria). Proc. Natl. Acad. Sci. USA 99, 11646–11651 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Culter, M.W. & Ward, W.W. Spectral Analysis and Proposed Model for GFP Dimerization. in Bioluminescence and Chemiluminescence: Molecular Reporting with Photons (eds. Hastings, J.W., Kriska, L.J., & Stanley, P.E.) 403–406 (Wiley & Sons, New York, 1997).

    Google Scholar 

  44. Campbell, R.E. et al. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877–7882 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Verkhusha, V.V. et al. High stability of Discosoma DsRed as compared to Aequorea EGFP. Biochemistry 42, 7879–7884 (2003).

    Article  CAS  PubMed  Google Scholar 

  46. Verkhusha, V.V. et al. Effect of high pressure and reversed micelles on the fluorescent proteins. Biochim. Biophys. Acta 1622, 192–195 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Verkhusha, V.V. et al. An enhanced mutant of red fluorescent protein DsRed for double labeling and developmental timer of neural fiber bundle formation. J. Biol. Chem. 276, 29621–29624 (2001).

    Article  CAS  PubMed  Google Scholar 

  48. Ward, W.W. Biochemical and Physical Properties of Green Fluorescent Protein. in Green Fluorescent Protein: Properties, Applications, and Protocols (eds. Chalfie, M. & Kain, S.) 45–75 (Wiley-Liss, New York, 1998).

    Google Scholar 

  49. Bevis, B.J. & Glick, B.S. Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat. Biotechnol. 20, 83–87 (2002).

    Article  CAS  PubMed  Google Scholar 

  50. Yanushevich, Y.G. et al. A strategy for the generation of non-aggregating mutants of Anthozoa fluorescent proteins. FEBS Lett. 511, 11–14 (2002).

    Article  CAS  PubMed  Google Scholar 

  51. Lauf, U. et al. Expression of fluorescently tagged connexins: a novel approach to rescue function of oligomeric DsRed-tagged proteins. FEBS Lett. 498, 11–15 (2001).

    Article  CAS  PubMed  Google Scholar 

  52. Karasawa S, et al. A green-emitting fluorescent protein from Galaxeidae coral and its monomeric version for use in fluorescent labeling. J. Biol. Chem. 278, 34167–34171 (2003).

    Article  CAS  PubMed  Google Scholar 

  53. Fradkov, A.F. et al. Far-red fluorescent tag for protein labelling. Biochem. J. 368, 17–21 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Gavin, P. et al. An approach for reducing unwanted oligomerisation of DsRed fusion proteins. Biochem. Biophys. Res. Commun. 298, 707–713 (2002).

    Article  CAS  PubMed  Google Scholar 

  55. Bulina, M.E. et al. Heterooligomeric tagging diminishes non-specific aggregation of target proteins fused with Anthozoa fluorescent proteins. Biochem. J. 371, 109–114 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Cotlet, M. et al. Identification of different emitting species in the red fluorescent protein DsRed by means of ensemble and single-molecule spectroscopy. Proc. Natl. Acad. Sci. USA 98, 14398–14403 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Garcia-Parajo, M.F. et al. The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection. Proc. Natl. Acad. Sci. USA 98, 14392–14397 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Mas, P. et al. Functional interaction of phytochrome B and cryptochrome 2. Nature 408, 207–211 (2000).

    Article  CAS  PubMed  Google Scholar 

  59. Hawley, T.S. et al. Four-color flow cytometric detection of retrovirally expressed red, yellow, green, and cyan fluorescent proteins. Biotechniques 30, 1028–1034 (2001).

    Article  CAS  PubMed  Google Scholar 

  60. Moede, T. et al. Online monitoring of stimulus-induced gene expression in pancreatic beta-cells. Diabetes 50, S15–S19 (2001).

    Article  CAS  PubMed  Google Scholar 

  61. Peloquin, J.J., Lauzon, C.R., Potter, S. & Miller, T.A. Transformed bacterial symbionts re-introduced to and detected in host gut. Curr. Microbiol. 45, 41–45 (2002).

    Article  CAS  PubMed  Google Scholar 

  62. Pollok, B.A. & Heim, R. Using GFP in FRET-based applications. Trends Cell Biol. 9, 57–60 (1999).

    Article  CAS  PubMed  Google Scholar 

  63. Miyawaki, A. & Tsien, R.Y. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 327, 472–500 (2000).

    Article  CAS  PubMed  Google Scholar 

  64. Patterson, G.H. et al. Forster distances between green fluorescent protein pairs. Anal. Biochem. 284, 438–440 (2000).

    Article  CAS  PubMed  Google Scholar 

  65. Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

    Article  CAS  PubMed  Google Scholar 

  66. Kohl, T. et al. A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins. Proc. Natl. Acad. Sci. USA 99, 12161–12166 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Adams, S.R. et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem. Soc. 124, 6063–6076 (2002).

    Article  CAS  PubMed  Google Scholar 

  68. Griffin, B.A. et al. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).

    Article  CAS  PubMed  Google Scholar 

  69. Xu, Y. et al. A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc. Natl. Acad. Sci. USA 96, 151–156 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Angers, S. et al. Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. USA 97, 3684–3689 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Reits, E.A. & Neefjes, J.J. From fixed to FRAP: measuring protein mobility and activity in living cells. Nat. Cell Biol. 3, 145–147 (2001).

    Article  Google Scholar 

  72. Lippincott-Schwartz, J. et al. Studying protein dynamics in living cells. Nat. Rev. Mol. Cell. Biol. 2, 444–456 (2001).

    Article  CAS  PubMed  Google Scholar 

  73. Marchant, J.S. et al. Multiphoton-evoked color change of DsRed as an optical highlighter for cellular and subcellular labeling. Nat. Biotechnol. 19, 645–649 (2001).

    Article  CAS  PubMed  Google Scholar 

  74. Patterson, G.H., & Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002).

    Article  CAS  PubMed  Google Scholar 

  75. Yokoe, H. & Meyer, T. Spatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement. Nat. Biotechnol. 14, 1252–1256 (1996).

    Article  CAS  PubMed  Google Scholar 

  76. Chudakov, D.M. et al. Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21, 191–194 (2003).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by grants from the National Institutes of Health (V.V.V.), European Office of Aerospace Research and Development under International Science and Technology Center partner project 2325 and Russian Academy of Sciences for the program “Physicochemical Biology” (K.A.L.).

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  1. Department of Pharmacology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, C236, Denver, 80262, Colorado, USA

    Vladislav V Verkhusha

  2. Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya 16/10, Moscow, 117997, Russia

    Konstantin A Lukyanov

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Correspondence to Vladislav V Verkhusha.

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Verkhusha, V., Lukyanov, K. The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nat Biotechnol 22, 289–296 (2004). https://doi.org/10.1038/nbt943

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