Cell and Tissue Biology

, Volume 11, Issue 1, pp 16–26 | Cite as

Design of near-infrared single-domain fluorescent protein GAF-FP based on bacterial phytochrome

  • K. A. Rumyantsev
  • D. M. Shcherbakova
  • N. I. Zakharova
  • V. V. Verkhusha
  • K. K. TuroverovEmail author


Fluorescent proteins (FPs) are widely used as genetically encoded markers for quantitative and noninvasive study of biological processes. Development of biomarkers that are fluorescent in the near-infrared spectral range allows the tissues of animals to be studied at a deeper level because they are more permeable to the light of this wavelength range than that of visible range. Such properties as low molecular weight and monomeric state are important for widespread use of FPs. In this paper, we managed to obtain FP based on the chromophore-binding domain of bacterial phytochrome (BphP) from Rhodopseudomonas palustris (RpB-phP1), named GAF-FP, with a molecular weight of ~19 kDa, which is half that of other FP based on BphP and 1.4 times lower than that of commonly used GFP-like proteins, which are fluorescent in the near-infrared range. In contrast to most other near-infrared FPs, GAF-FP is a monomer, which has a high photostability, and its structure is stable to the incorporation of small peptide inserts. Moreover, GAF-FP is capable of covalent attachment of two different tetrapyrrole chromophores: phycocyanobilin (PCB) and biliverdin (BV), which is contained in mammalian tissues. GAF-FP with attached BV as a chromophore (GAF-FP–BV) has the main absorption band with a maximum at 635 nm. The fluorescence maximum falls at 670 nm, whereby GAF-FP has a high ratio of the fluorescence signal to the background signal even if FP is localized at a depth of several mm below the tissue surface. Together with the near-infrared absorption band, GAF-FP–BV also has an absorption band in the violet region of the spectrum with a maximum at 378 nm. We used this property to design a chimeric protein consisting of modified luciferase from Renilla reniformis (RLuc8) and GAF-FP. We showed resonance energy transfer from the substrate, the excited state of which occurs when oxidized by luciferase, to the chromophore GAF-FP–BV in the designed fusion protein. In the absence of an energy acceptor, RLuc8 catalyzes the cleavage of the substrate with the emission of the light with a maximum at 400 nm. At the same time, the energy from the substrate is transferred to the FP chromophore and then emitted in the near-infrared range corresponding to the spectrum of GAF-FP fluorescence in the GAF-FP–RLuc8 chimeric protein. These results open the way for the development of new small near-infrared FPs based on various natural BphPs with a view to their widespread use in cell and molecular biology.


monomerism fluorescent proteins phytochromes bioluminescence 



bacterial phytochrome


biliverdin IXa




fluorescent protein


green fluorescent protein


Renilla reniformis luciferase


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Auldridge, M.E., Satyshur, K.A., Anstrom, D.M, and Forest, K.T., Structure-guided engineering enhances a phytochrome- based infrared fluorescent protein, J. Biol. Chem., 2012, vol. 287, pp. 7000–7009.CrossRefPubMedGoogle Scholar
  2. Bellini, D. and Papiz, M.Z., Structure of a bacteriophytochrome and light-stimulated protomer swapping with a gene repressor, Structure, 2012, vol. 20, pp. 1436–1446.CrossRefPubMedGoogle Scholar
  3. Bhattacharya, S., Auldridge, M.E., Lehtivuori, H., Ihalainen, J.A., and Forest, K.T., Origins of fluorescence in evolved bacteriophytochromes, J. Biol. Chem., 2014, vol. 289, pp. 32144–32152.CrossRefPubMedPubMedCentralGoogle Scholar
  4. Bhoo, S.H., Davis, S.J., Walker, J., Karniol, B., and Vierstra, R.D., Bacteriophytochromes are photochromic histidine kinases using a biliverdin chromophore, Nature, 2001, vol. 414, pp. 776–779.CrossRefPubMedGoogle Scholar
  5. Chen, M., Li, W., Zhang, Z., Liu, S., Zhang, X., Zhang, X.E., and Cui, Z., Novel near-infrared BiFC systems from a bacterial phytochrome for imaging protein interactions and drug evaluation under physiological conditions, Biomaterials, 2015, vol. 48, pp. 97–107.CrossRefPubMedGoogle Scholar
  6. Filonov, G.S., Piatkevich, K.D., Ting, L.M., Zhang, J., Kim, K., and Verkhusha, V.V., Bright and stable near-infrared fluorescent protein for in vivo imaging, Nat. Biotechnol., 2011, vol. 29, pp. 757–761.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Filonov, G.S. and Verkhusha, V.V., A near-infrared bifc reporter for in vivo imaging of protein-protein interactions, Chem. Biol., 2013, vol. 20, pp. 1078–1086.CrossRefPubMedPubMedCentralGoogle Scholar
  8. Fischer, A.J., Rockwell, N.C., Jang, A.Y., Ernst, L.A., Waggoner, A.S., Duan, Y., Lei, H., and Lagarias, J.C., Multiple roles of a conserved GAF domain tyrosine residue in cyanobacterial and plant phytochromes, Biochemistry, 2005, vol. 44, pp. 15203–15215.CrossRefPubMedPubMedCentralGoogle Scholar
  9. Gambetta, G.A. and Lagarias, J.C., Genetic engineering of phytochrome biosynthesis in bacteria, Proc. Natl. Acad. Sci. U. S. A., 2001, vol. 98, pp. 10566–10571.CrossRefPubMedPubMedCentralGoogle Scholar
  10. Ishizuka, T., Narikawa, R., Kohchi, T., Katayama, M., and Ikeuchi, M., Cyanobacteriochrome TePixJ of Thermosyn-echococcus elongatus harbors phycoviolobilin as a chromophore, Plant Cell Physiol., 2007, vol. 48, pp. 1385–1390.CrossRefPubMedGoogle Scholar
  11. Lamparter, T., Evolution of cyanobacterial and plant phytochromes, FEBS Lett., 2004, vol. 573, pp. 1–5.CrossRefPubMedGoogle Scholar
  12. Narikawa, R., Fukushima, Y., Ishizuka, T., Itoh, S., and Ikeuchi, M., A novel photoactive GAF domain of cyanobacteriochrome AnPixJ that shows reversible green/red photoconversion, J. Mol. Biol., 2008, vol. 380, pp. 844–855.CrossRefPubMedGoogle Scholar
  13. Narikawa, R., Nakajima, T., Aono, Y., Fushimi, K., Enomoto, G., Ni, W., Itoh, S., Sato, M., and Ikeuchi, M., A biliverdin-binding cyanobacteriochrome from the chlorophyll D-bearing cyanobacterium Acaryochloris marina, Sci. Rep., 2015, vol. 5, p. 7950.CrossRefPubMedPubMedCentralGoogle Scholar
  14. Pedelacq, J.D., Cabantous, S., Tran, T., Terwilliger, T.C., and Waldo, G.S., Engineering and characterization of a superfolder green fluorescent protein, Nat. Biotechnol., 2006, vol. 24, pp. 79–88.CrossRefPubMedGoogle Scholar
  15. Piatkevich, K.D., Subach, F.V., and Verkhusha, V.V., Engineering of bacterial phytochromes for near-infrared imaging, sensing, and light-control in mammals, Chem. Soc. Rev., 2013a, vol. 42, pp. 3441–3452.PubMedGoogle Scholar
  16. Piatkevich, K.D., Subach, F.V., and Verkhusha, V.V., Farred light photoactivatable near-infrared fluorescent proteins engineered from a bacterial phytochrome, Nat. Commun., 2013b, vol. 4, p. 2153.CrossRefPubMedPubMedCentralGoogle Scholar
  17. Rockwell, N.C. and Lagarias, J.C., A brief history of phytochromes, Eur. J. Chem. Phys. Phys. Chem., 2010, vol. 11, pp. 1172–1180.Google Scholar
  18. Rockwell, N.C., Martin, S.S., and Lagarias, J.C., Red/green cyanobacteriochromes: sensors of color and power, Biochemistry, 2012, vol. 51, pp. 9667–9677.CrossRefPubMedGoogle Scholar
  19. Saito, K., Chang, Y.F., Horikawa, K., Hatsugai, N., Higuchi, Y., Hashida, M., Yoshida, Y., Matsuda, T., Arai, Y., and Nagai, T., Luminescent proteins for high-speed singlecell and whole-body imaging, Nat. Commun., 2012, vol. 3, pp. 1262.CrossRefPubMedPubMedCentralGoogle Scholar
  20. Schneider, C.A., Rasband, W.S., and Eliceiri, K.W., NIH image to ImageJ: 25 years of image analysis, Nat. Methods, 2012, vol. 9, pp. 671–675.CrossRefPubMedGoogle Scholar
  21. Shcherbakova, D.M. and Verkhusha, V.V., Near-infrared fluorescent proteins for multicolor in vivo imaging, Nat. Methods, 2013, vol. 10, pp. 751–754.CrossRefPubMedPubMedCentralGoogle Scholar
  22. Shcherbakova, D.M., Shemetov, A.A., Kaberniuk, A.A., and Verkhusha, V.V., Natural photoreceptors as a source of fluorescent proteins, biosensors, and optogenetic tools, Ann. Rev. Biochem., 2015, vol. 84, pp. 519–550.CrossRefPubMedGoogle Scholar
  23. Shu, X., Royant, A., Lin, M.Z., Aguilera, T.A., Lev-Ram, V., Steinbach, P.A., and Tsien, R.Y., Mammalian expression of infrared fluorescent proteins engineered from a bacterial phytochrome, Science, 2009, vol. 324, pp. 804–807.CrossRefPubMedPubMedCentralGoogle Scholar
  24. Stepanenko, O.V., Bublikov, G.S., Stepanenko, O.V., Shcherbakova, D.M., Verkhusha, V.V., Turoverov, K.K., and Kuznetsova, I.M., A knot in the protein structure— probing the near-infrared fluorescent protein iRFP designed from a bacterial phytochrome, FEBS J., 2014, vol. 281, pp. 2284–2298.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Takai, A., Nakano, M., Saito, K., Haruno, R., Watanabe, T.M., Ohyanagi, T., Jin, T., Okada, Y., and Nagai, T., Expanded palette of nano-lanterns for real-time multicolor luminescence imaging, Proc. Natl. Acad. Sci. USA, 2015, vol. 112, pp. 4352–4356.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Toh, K.C., Stojkovic, E.A., van Stokkum, I.H., Moffat, K., and Kennis, J.T., Fluorescence quantum yield and photochemistry of bacteriophytochrome constructs, Phys. Chem. Chem. Phys., 2011, vol. 13, pp. 11985–11997.CrossRefPubMedGoogle Scholar
  27. Wagner, J.R., Zhang, J., Brunzelle, J.S., Vierstra, R.D., and Forest, K.T., High resolution structure of Deinococcus bacteriophytochrome yields new insights into phytochrome architecture and evolution, J. Biol. Chem., 2007, vol. 282, pp. 12298–12309.CrossRefPubMedGoogle Scholar
  28. Yu, D., Gustafson, W.C., Han, C., Lafaye, C., Noirclerc-Savoye, M., Ge, W.P., Thayer, D.A., Huang, H., Kornberg, T.B., Royant, A., Jan, L.Y., Jan, Y.N., Weiss, W.A., and Shu, X., An improved monomeric infrared fluorescent protein for neuronal and tumour brain imaging, Nat. Commun., 2014, vol. 5, pp. 3626.PubMedPubMedCentralGoogle Scholar
  29. Zhang, J., Wu, X.J., Wang, Z.B., Chen, Y., Wang, X., Zhou, M., Scheer, H., and Zhao, K.H., Fused-gene approach to photoswitchable and fluorescent biliproteins, Angew. Chem., 2010, vol. 49, pp. 5456–5458.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2017

Authors and Affiliations

  • K. A. Rumyantsev
    • 1
    • 2
  • D. M. Shcherbakova
    • 2
  • N. I. Zakharova
    • 2
  • V. V. Verkhusha
    • 2
  • K. K. Turoverov
    • 1
    Email author
  1. 1.Institute of CytologyRussian Academy of SciencesSt. PetersburgRussia
  2. 2.Albert Einstein College of MedicineNew YorkUSA

Personalised recommendations