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Acta Biologica Hungarica

, Volume 57, Issue 2, pp 181–190 | Cite as

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

  • M. SzécsiEmail author
  • Margarethe Spindler-Barth
Article

Abstract

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.

Keywords

Fluorescent dyes site specific labeling recombinant fusion proteins 

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References

  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.CrossRefGoogle 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.CrossRefGoogle Scholar
  3. 3.
    Combet, C., Blanchet, C., Geourjon, C., Deleage, G. (2000) NPS@: Network protein sequence analysis. Trends Biochem. Sci. 25, 147–150.CrossRefGoogle 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.CrossRefGoogle Scholar
  5. 5.
    Geourjon, C., Deleage, G. (1994) SOPM: a self-optimized method for protein secondary structure prediction. Protein Eng. 7, 157–164.CrossRefGoogle 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.PubMedGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle Scholar
  11. 11.
    Hartley, J., Temple, G., Brasch, M. A. (2000) DNA cloning using in vitro site-specific recombination. Genome Research 10, 1788–1795.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle Scholar
  21. 21.
    Riddiford, L. M., Cherbas, P., Truman, J. W. (2000) Ecdysone receptors and their biological actions. Vitam. Horm. 60, 1–73.CrossRefGoogle 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.CrossRefGoogle Scholar
  23. 23.
    Spindler, K. D., Przibilla, S., Spindler-Barth, M. (2001) Moulting hormones of arthropods: Molecular mechanisms. Zoology 103, 189–201.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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle 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.CrossRefGoogle Scholar

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© Akadémiai Kiadó, Budapest 2006

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  1. 1.Department of General Zoology and EndocrinologyUniversity of UlmUlmGermany

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