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.
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.
'New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applications ' () 124 J. Am. Chem. Soc. : 6063 -6076 .
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.
'Genetically targeted chro-mophore-assisted light inactivation ' () 21 Nat. Biotechnol. : 1505 -1508 .
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. Nature336, 476-479.
'Functional ecdysone receptor is the product of ecr and usp genes ' () 336 Nature : 476 -479 .
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.
'A new protein conformation indicator based on biarsenical fluorescein with an extended benzoic acid moiety ' () 20 Anal. Sci. : 273 -278 .
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.
'Polarity of the ecdysone receptor complex interaction with the palindromic response element from the hsp27 gene promoter ' () 267 Eur. J. Biochem. : 507 -519 .
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.
'An analysis of ecdysone receptor domains required for heterodimerization with ultraspiracle ' () 41 Arch. Insect Biochem. Physiol. : 61 -70 .
Riddiford, L. M., Cherbas, P., Truman, J. W. (2000) Ecdysone receptors and their biological actions. Vitam. Horm.60, 1-73.
'Ecdysone receptors and their biological actions ' () 60 Vitam. Horm. : 1 -73 .
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 biarsenical ligands as a tool for investigating gap junction structure and dynamics. Cell Commun. Adhes.10, 181-186.
'Tetracysteine genetic tags complexed with biarsenical ligands as a tool for investigating gap junction structure and dynamics ' () 10 Cell Commun. Adhes. : 181 -186 .
Spindler, K. D., Przibilla, S., Spindler-Barth, M. (2001) Moulting hormones of arthropods: Molecular mechanisms. Zoology103, 189-201.
'Moulting hormones of arthropods: Molecular mechanisms ' () 103 Zoology : 189 -201 .
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.
'The protein-labeling reagent FlAsH-EDT2 binds not only to CCXXCC motifs but also non-specifically to endogenous cysteine-rich proteins ' () 442 Eur. J. Physiol. : 859 -866 .
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.
'A novel method of affinity-purifying proteins using bis-arsenical fluorescein ' () 9 Protein Sci. : 213 -217 .
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.
'Characterization of the ligand-binding domain of the ecdysteroid receptor from Drosophila melanogaster ' () 384 Biol. Chem. : 105 -116 .
Griffin, B. A., Adams, S. R., Tsien, R. Y. (1998) Specific covalent labeling of recombinant protein molecules inside live cells. Science281, 269-281.
'Specific covalent labeling of recombinant protein molecules inside live cells ' () 281 Science : 269 -281 .
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.
'Fluorescent labeling of recombinant proteins in living cells with FlAsH ' () 327 Methods Enzymol. : 565 -578 .
Marek, K. W., Davis, G. W. (2002) Transgenetically encoded protein photoinactivation (FlAsH-FALI): acute inactivation of synaptotagmin I. Neuron36, 805-813.
'Transgenetically encoded protein photoinactivation (FlAsH-FALI): acute inactivation of synaptotagmin I ' () 36 Neuron : 805 -813 .
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.
'Imaging of con-formational changes of proteins with new environment-sensitive fluorescent probe designed for site specific labeling of recombinant proteins in live cells ' () 73 Anal. Chem. : 2920 -2928 .
Hartley, J., Temple, G., Brasch, M. A. (2000) DNA cloning using in vitro site-specific recombination. Genome Research10, 1788-1795.
'DNA cloning using in vitro site-specific recombination ' () 10 Genome Research : 1788 -1795 .
Ignatova, Z., Gierasch, L. M. (2004) Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling. Proc. Natl. Acad. Sci. USA101, 523-528.
'Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling ' () 101 Proc. Natl. Acad. Sci. USA : 523 -528 .
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.
'Activity-dependent regulation of dendritic synthesis and trafficking of AMPA receptors ' () 7 Nat. Neurosci. : 244 -253 .
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.
'Ligand induced heterodimerization between ligand binding domains of Drosophila ecdysteroid receptor and ultraspiracle ' () 269 Eur. J. Biochem. : 3237 -3245 .
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.
'A peptide C-terminal to the second Zn finger of human vitamin D receptor is able to specify nuclear localization ' () 223 Eur. J. Biochem. : 381 -387 .
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.
'Crystal structure of the ligand binding domain of the ultraspiracle protein USP, the ortholog of RXRs in insects ' () 276 J. Biol. Chem. : 7465 -7474 .
Combet, C., Blanchet, C., Geourjon, C., Deleage, G. (2000) NPS@: Network protein sequence analysis. Trends Biochem. Sci.25, 147-150.
'NPS@: Network protein sequence analysis ' () 25 Trends Biochem. Sci. : 147 -150 .
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. Science296, 503-507.
'Multicolor and electron microscopic imaging of connexin trafficing ' () 296 Science : 503 -507 .
Geourjon, C., Deleage, G. (1994) SOPM: a self-optimized method for protein secondary structure prediction. Protein Eng.7, 157-164.
'SOPM: a self-optimized method for protein secondary structure prediction ' () 7 Protein Eng. : 157 -164 .
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.
'SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments ' () 11 Comput. Appl. Biosci. : 681 -684 .
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.
'Analysis of Usp DNA binding domain targeting reveals critical determinants of the ecdysone receptor complex interaction with the response element ' () 268 Eur. J. Biochem. : 3751 -3758 .