FlAsH Labeling of A Nuclear Receptor Domain (D Domain of Ultraspiracle) Fused to Tetracysteine Tag


Biarsenical fluorescein compounds feature unique fluorescence characteristics and special binding mechanism to tetracysteine tags with certain structures and these dyes offer a feasible method for site specific labeling of heterologously expressed proteins. We aimed FlAsH fluorescent labeling of tetracysteine fused hinge region of the ultraspiracle from Drosophila melanogaster (DmUSP-D domain) to facilitate functional studies of this receptor domain. A CCPGCC tetracysteine motif was integrated between His6, Gateway attB1, and Flag tags and attached to the N-terminus of the DmUSP-D. The fusion protein was expressed in Esherichia coli and the FlAsH labeling was performed in bacterial extracts, under conditions which are compatible with receptor function. The dye was bound to the tetracysteine tag with high affinity and complex stability and the labeling proved to be specific for the target fusion protein. Results indicate that FlAsH labeling of the internal CCPGCC motif can be a valuable tool for the functional characterisation of any nuclear receptor domains.


  1. 1.

    Adams, S. R., Cambell, R. E., Gross, L. A., Martin, B. R., Walkup, G. K., Yao, Y., Llopis, J., Tsien, R. Y. (2002) 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.

    CAS  Article  Google Scholar 

  2. 2.

    Billas, I. M., Moulinier, L., Rochel, N., Moras, D. (2001) Crystal structure of the ligand binding domain of the ultraspiracle protein USP, the ortholog of RXRs in insects. J. Biol. Chem. 276, 7465–7474.

    CAS  Article  Google Scholar 

  3. 3.

    Combet, C., Blanchet, C., Geourjon, C., Deleage, G. (2000) NPS@: Network protein sequence analysis. Trends Biochem. Sci. 25, 147–150.

    CAS  Article  Google Scholar 

  4. 4.

    Gaietta, G., Deerinck, T. J., Adams, S. R., Bouwer, J., Tour, O., Laird, D. W., Sosinsky, G. E., Tsien, R. Y., Ellisman, M. H. (2002) Multicolor and electron microscopic imaging of connexin trafficing. Science 296, 503–507.

    CAS  Article  Google Scholar 

  5. 5.

    Geourjon, C., Deleage, G. (1994) SOPM: a self-optimized method for protein secondary structure prediction. Protein Eng. 7, 157–164.

    CAS  Article  Google Scholar 

  6. 6.

    Geourjon, C., Deleage, G. (1995) SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput. Appl. Biosci. 11, 681–684.

    CAS  PubMed  Google Scholar 

  7. 7.

    Grad, I., Niedziela-Majka, A., Kochman, M., Ozyhar, A. (2001) Analysis of Usp DNA binding domain targeting reveals critical determinants of the ecdysone receptor complex interaction with the response element. Eur. J. Biochem. 268, 3751–3758.

    CAS  Article  Google Scholar 

  8. 8.

    Grebe, M., Przibilla, S., Henrich, V. C., Spindler-Barth, M. (2003) Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster. Biol. Chem. 384, 105–116.

    CAS  Article  Google Scholar 

  9. 9.

    Griffin, B. A., Adams, S. R., Tsien, R. Y. (1998) Specific covalent labeling of recombinant protein molecules inside live cells. Science 281, 269–281.

    CAS  Article  Google Scholar 

  10. 10.

    Griffin, B. A., Adams, S. R., Jones, J., Tsien, R. Y. (2000) Fluorescent labeling of recombinant proteins in living cells with FlAsH. Methods Enzymol. 327, 565–578.

    CAS  Article  Google Scholar 

  11. 11.

    Hartley, J., Temple, G., Brasch, M. A. (2000) DNA cloning using in vitro site-specific recombination. Genome Research 10, 1788–1795.

    CAS  Article  Google Scholar 

  12. 12.

    Ignatova, Z., Gierasch, L. M. (2004) Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling. Proc. Natl. Acad. Sci. USA 101, 523–528.

    CAS  Article  Google Scholar 

  13. 13.

    Ju, W., Morishita, W., Tsui, J., Gaietta, G., Deerinck, T. J., Adams, S. R., Garner, C. C., Tsien, R. Y., Ellisman, M. H., Malenka, R. C. (2004) Activity-dependent regulation of dendritic synthesis and trafficking of AMPA receptors. Nat. Neurosci. 7, 244–253.

    CAS  Article  Google Scholar 

  14. 14.

    Lezzi, M., Bergman, T., Henrich, V. C., Vogtli, M., Fromel, C., Grebe, M., Przibilla, S., Spindler-Barth, M. (2002) Ligand induced heterodimerization between ligand binding domains of Drosophila ecdysteroid receptor and ultraspiracle. Eur. J. Biochem. 269, 3237–3245.

    CAS  Article  Google Scholar 

  15. 15.

    Luo, Z., Rouvinen, J., Maenpaa, P. H. (1994) A peptide C-terminal to the second Zn finger of human vitamin D receptor is able to specify nuclear localization. Eur. J. Biochem. 223, 381–387.

    CAS  Article  Google Scholar 

  16. 16.

    Marek, K. W., Davis, G. W. (2002) Transgenetically encoded protein photoinactivation (FlAsH-FALI): acute inactivation of synaptotagmin I. Neuron 36, 805–813.

    CAS  Article  Google Scholar 

  17. 17.

    Nakanishi, J., Nakajima, T., Sato, M., Ozawa, T., Tohda, K., Umezawa, Y. (2001) Imaging of con-formational changes of proteins with new environment-sensitive fluorescent probe designed for site specific labeling of recombinant proteins in live cells. Anal. Chem. 73, 2920–2928.

    CAS  Article  Google Scholar 

  18. 18.

    Nakanishi, J., Maeda, M., Umezawa, Y. (2004) A new protein conformation indicator based on biarsenical fluorescein with an extended benzoic acid moiety. Anal. Sci. 20, 273–278.

    CAS  Article  Google Scholar 

  19. 19.

    Niedziela-Majka, A., Kochman, M., Ozyhar, A. (2000) Polarity of the ecdysone receptor complex interaction with the palindromic response element from the hsp27 gene promoter. Eur. J. Biochem. 267, 507–519.

    CAS  Article  Google Scholar 

  20. 20.

    Pereira, S. C., Sundaram, M., Krell, P. J., Retnakaran, A., Dhadialla, T. S., Palli, S. R. (1999) An analysis of ecdysone receptor domains required for heterodimerization with ultraspiracle. Arch. Insect Biochem. Physiol. 41, 61–70.

    Article  Google Scholar 

  21. 21.

    Riddiford, L. M., Cherbas, P., Truman, J. W. (2000) Ecdysone receptors and their biological actions. Vitam. Horm. 60, 1–73.

    CAS  Article  Google Scholar 

  22. 22.

    Sosinsky, G. E., Gaietta, G. M., Hand, G., Deerinck, T. J., Han, A., Mackey, M., Adams, S. R., Bouwer, J., Tsien, R. Y., Ellisman, M. H. (2003) Tetracysteine genetic tags complexed with biarseni-cal ligands as a tool for investigating gap junction structure and dynamics. Cell Commun. Adhes. 10, 181–186.

    CAS  Article  Google Scholar 

  23. 23.

    Spindler, K. D., Przibilla, S., Spindler-Barth, M. (2001) Moulting hormones of arthropods: Molecular mechanisms. Zoology 103, 189–201.

    CAS  Google Scholar 

  24. 24.

    Stroffekova, K., Proenza, C., Beam, K. G. (2001) The protein-labeling reagent FlAsH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins. Eur. J. Physiol. 442, 859–866.

    CAS  Article  Google Scholar 

  25. 25.

    Thorn, K. S., Naber, N., Matuska, M., Vale, R. D., Cooke, R. (2000) A novel method of affinity-purifying proteins using bis-arsenical fluorescein. Protein Sci. 9, 213–217.

    CAS  Article  Google Scholar 

  26. 26.

    Tour, O., Meijer, R. M., Zacharias, D. A., Adams, S. R., Tsien, R. Y. (2003) Genetically targeted chro-mophore-assisted light inactivation. Nat. Biotechnol. 21, 1505–1508.

    CAS  Article  Google Scholar 

  27. 27.

    Yao, T. P., Forman, B. M., Jiang, Z., Cherbas, L., Chen, J. D., McKewon, M., Cherbas, P., Evans, R. M. (1993) Functional ecdysone receptor is the product of ecr and usp genes. Nature 336, 476–479.

    Article  Google Scholar 

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Correspondence to M. Szécsi.

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Szécsi, M., Spindler-Barth, M. FlAsH Labeling of A Nuclear Receptor Domain (D Domain of Ultraspiracle) Fused to Tetracysteine Tag. BIOLOGIA FUTURA 57, 181–190 (2006). https://doi.org/10.1556/ABiol.57.2006.2.5

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  • Fluorescent dyes
  • site specific labeling
  • recombinant fusion proteins