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.
Subscribe to Journal
Get full journal access for 1 year
only $20.83 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Prasher, D.C. et al. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229–233 (1992).
Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).
Matz, M.V. et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol. 17, 969–973 (1999).
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).
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).
Labas, Y.A. et al. Diversity and evolution of the green fluorescent protein family. Proc. Natl. Acad. Sci. USA 99, 4256–4261 (2002).
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).
Gurskaya, N.G. et al. GFP-like chromoproteins as a source of far-red fluorescent proteins. FEBS Lett. 507, 16–20 (2001).
Schlichter, D. et al. Light harvesting by wavelength transformation in symbiotic coral of the Red Sea twilight zone. Mar. Biol. 91, 403–407 (1986).
Salih, A. et al. Fluorescent pigments in corals are photoprotective. Nature 408, 850–853 (2000).
Wilson. T. & Hastings, J.W. Bioluminescence. Annu. Rev. Cell Dev. Biol. 14, 197–230 (1998).
Heim, R. et al. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 91, 12501–12504 (1994).
Chalfie, M. et al. Green fluorescent protein as a marker for gene expression. Science 263, 802–805 (1994).
Cubitt, A.B. et al. Understanding, improving and using green fluorescent proteins. Trends Biochem. Sci. 20, 448–455 (1995).
Fukuda, H. et al. Folding of green fluorescent protein and the cycle3 mutant. Biochemistry 39, 12025–12032 (2000).
Zhang, J. et al. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell. Biol. 3, 906–918 (2002).
Lippincott-Schwartz, J. & Patterson, G.H. Development and use of fluorescent protein markers in living cells. Science 300, 87–91 (2003).
Zimmer, M. Green fluorescent protein: applications, structure, and related photophysical behavior. Chem. Rev. 102, 759–781 (2002).
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).
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).
Remington, S.J. Structural basis for understanding spectral variations in green fluorescent protein. Methods Enzymol. 305, 196–211 (2000).
Wall, M.A. et al. The structural basis for red fluorescence in the tetrameric GFP homolog DsRed. Nat. Struct. Biol. 7, 1133–1138 (2000).
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).
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).
Prescott, M. et al. The 2.2 Å crystal structure of a pocilloporin pigment reveals a nonplanar chromophore conformation. Structure 11, 275–284 (2003).
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).
Chudakov, D.M. et al. Chromophore environment provides clue to “kindling fluorescent protein” riddle. J. Biol. Chem. 278, 7215–7219 (2003).
Mizuno, H. et al. Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein. Molecular Cell 12, 1051–1058 (2003).
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).
Terskikh, A. et al. “Fluorescent timer”: protein that changes color with time. Science 290, 1585–1588 (2000).
Wiehler, J. et al. Mutants of Discosoma red fluorescent protein with a GFP-like chromophore. FEBS Lett. 487, 384–389 (2001).
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).
Terskikh, A.V. et al. Analysis of DsRed mutants. Space around the fluorophore accelerates fluorescence development. J. Biol. Chem. 277, 7633–7636 (2002).
Gurskaya, N.G. et al. Color transitions in coral's fluorescent proteins by site-directed mutagenesis. BMC Biochem. 2, 6 (2001).
Bulina, M.E. et al. Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis. BMC Biochem. 3, 7 (2002).
Duncan, R.R. et al. Functional and spatial segregation of secretory vesicle pools according to vesicle age. Nature 422, 176–180 (2003).
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).
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).
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).
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).
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).
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).
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).
Campbell, R.E. et al. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877–7882 (2002).
Verkhusha, V.V. et al. High stability of Discosoma DsRed as compared to Aequorea EGFP. Biochemistry 42, 7879–7884 (2003).
Verkhusha, V.V. et al. Effect of high pressure and reversed micelles on the fluorescent proteins. Biochim. Biophys. Acta 1622, 192–195 (2003).
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).
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).
Bevis, B.J. & Glick, B.S. Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat. Biotechnol. 20, 83–87 (2002).
Yanushevich, Y.G. et al. A strategy for the generation of non-aggregating mutants of Anthozoa fluorescent proteins. FEBS Lett. 511, 11–14 (2002).
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).
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).
Fradkov, A.F. et al. Far-red fluorescent tag for protein labelling. Biochem. J. 368, 17–21 (2002).
Gavin, P. et al. An approach for reducing unwanted oligomerisation of DsRed fusion proteins. Biochem. Biophys. Res. Commun. 298, 707–713 (2002).
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).
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).
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).
Mas, P. et al. Functional interaction of phytochrome B and cryptochrome 2. Nature 408, 207–211 (2000).
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).
Moede, T. et al. Online monitoring of stimulus-induced gene expression in pancreatic beta-cells. Diabetes 50, S15–S19 (2001).
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).
Pollok, B.A. & Heim, R. Using GFP in FRET-based applications. Trends Cell Biol. 9, 57–60 (1999).
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).
Patterson, G.H. et al. Forster distances between green fluorescent protein pairs. Anal. Biochem. 284, 438–440 (2000).
Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).
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).
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).
Griffin, B.A. et al. Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–272 (1998).
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).
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).
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).
Lippincott-Schwartz, J. et al. Studying protein dynamics in living cells. Nat. Rev. Mol. Cell. Biol. 2, 444–456 (2001).
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).
Patterson, G.H., & Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–1877 (2002).
Yokoe, H. & Meyer, T. Spatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement. Nat. Biotechnol. 14, 1252–1256 (1996).
Chudakov, D.M. et al. Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21, 191–194 (2003).
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.).
The authors declare no competing financial interests.
About this article
Cite this article
Verkhusha, V., Lukyanov, K. The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins. Nat Biotechnol 22, 289–296 (2004) doi:10.1038/nbt943
Evaluation of in vitro and in vivo antibiotic efficacy against a novel bioluminescent Shigella flexneri
Scientific Reports (2019)
Design of a chromogenic substrate for elastase based on split GFP system—Proof of concept for colour switch sensors
Biotechnology Reports (2019)
Highly tunable multiple narrow emissions of dyed dielectric-metal core–shell resonators: towards efficient fluorescent labels
Crystal structure of the blue fluorescent protein with a Leu-Leu-Gly tri-peptide chromophore derived from the purple chromoprotein of Stichodactyla haddoni
International Journal of Biological Macromolecules (2019)
Polyglutamine toxicity assays highlight the advantages of mScarlet for imaging in Saccharomyces cerevisiae