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Coelenterazine-v ligated to Ca2+-triggered coelenterazine-binding protein is a stable and efficient substrate of the red-shifted mutant of Renilla muelleri luciferase

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Abstract

It has been shown that the coelenterazine analog, coelenterazine-v, is an efficient substrate for a reaction catalyzed by Renilla luciferase. The resulting bioluminescence emission maximum is shifted to a longer wavelength up to 40 nm, which allows the use of some “yellow” Renilla luciferase mutants for in vivo imaging. However, the utility of coelenterazine-v in small-animal imaging has been hampered by its instability in solution and in biological tissues. To overcome this drawback, we ligated coelenterazine-v to Ca2+-triggered coelenterazine-binding protein from Renilla muelleri, which apparently functions in the organism for stabilizing and protecting coelenterazine from oxidation. The coelenterazine-v bound within coelenterazine-binding protein has revealed a greater long-term stability at both 4 and 37 °C. In addition, the coelenterazine-binding protein ligated by coelenterazine-v yields twice the total light over free coelenterazine-v as a substrate for the red-shifted R. muelleri luciferase. These findings suggest the possibility for effective application of coelenterazine-v in various in vitro assays.

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Abbreviations

CBP:

Ca2+-triggered coelenterazine-binding protein

RM-Luc:

Native Renilla muelleri luciferase

RM-Y:

Long-wavelength shifted Renilla muelleri luciferase mutant

References

  1. Cormier MJ, Lee J, Wampler JE (1975) Bioluminescence. Recent advances. Annu Rev Biochem 44:255–272

    Article  CAS  Google Scholar 

  2. Hori K, Wampler JE, Matthews JC, Cormier MJ (1973) Identification of the product excited states during the chemiluminescent and bioluminescent oxidation of Renilla (sea pansy) luciferin and certain of its analogs. Biochemistry 12:4463–4468

    Article  CAS  Google Scholar 

  3. Ward WW, Cormier MJ (1979) An energy transfer protein in coelenterate bioluminescence. Characterization of the Renilla green-fluorescent protein. J Biol Chem 254:781–788

    CAS  Google Scholar 

  4. Charbonneau H, Cormier MJ (1979) Ca2+-induced bioluminescence in Renilla reniformis. Purification and characterization of a calcium-triggered luciferin-binding protein. J Biol Chem 254:769–780

    CAS  Google Scholar 

  5. Kumar S, Harrylock M, Walsh KA, Cormier MJ, Charbonneau H (1990) Amino acid sequence of the Ca2+-triggered luciferin binding protein of Renilla reniformis. FEBS Lett 268:287–290

    Article  CAS  Google Scholar 

  6. Moncrief ND, Kretsinger RH, Goodman M (1990) Evolution of EF-hand calcium-modulated proteins. I. Relationships based on amino acid sequences. J Mol Evol 30:522–562

    Article  CAS  Google Scholar 

  7. Kawasaki H, Nakayama S, Kretsinger RH (1998) Classification and evolution of EF-hand proteins. Biometals 11:277–295

    Article  CAS  Google Scholar 

  8. Cormier MJ (1978) Applications of Renilla bioluminescence: an introduction. Methods Enzymol 57:237–244

    Article  CAS  Google Scholar 

  9. Inoyue S (2007) Expression, purification and characterization of calcium-triggered luciferin-binding protein of Renilla reniformis. Protein Expr Purif 52:66–73

    Article  Google Scholar 

  10. Titushin MS, Markova SV, Frank LA, Malikova NP, Stepanyuk GA, Lee J, Vysotski ES (2008) Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase. Photochem Photobiol Sci 7:189–196

    Article  CAS  Google Scholar 

  11. Stepanyuk GA, Liu ZJ, Markova SV, Frank LA, Lee J, Vysotski ES, Wang BC (2008) Crystal structure of coelenterazine-binding protein from Renilla muelleri at 1.7 Å: why it is not a calcium-regulated photoprotein. Photochem Photobiol Sci 7:442–447

    Article  CAS  Google Scholar 

  12. Stepanyuk GA, Liu ZJ, Vysotski ES, Lee J, Rose JP, Wang BC (2009) Structure based mechanism of the Ca2+-induced release of coelenterazine from the Renilla binding protein. Proteins 74:583–593

    Article  CAS  Google Scholar 

  13. Vysotski ES, Lee J (2004) Ca2+-regulated photoproteins: structural insight into the bioluminescence mechanism. Acc Chem Res 37:405–415

    Article  CAS  Google Scholar 

  14. Shimomura O (2006) Bioluminescence: chemical principles and methods. World Scientific, Singapore

    Book  Google Scholar 

  15. de Wet JR, Wood KV, Helinski DR, DeLuca M (1985) Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proc Natl Acad Sci USA 82:7870–7873

    Article  Google Scholar 

  16. Contag CH, Spilman SD, Contag PR, Oshiro M, Eames B, Dennery P, Stevenson DK, Benaron DA (1997) Visualizing gene expression in living mammals using a bioluminescent reporter. Photochem Photobiol 66:523–531

    Article  CAS  Google Scholar 

  17. Yegutkin GG, Samburski SS, Jalkanen S (2003) Soluble purine-converting enzymes circulate in human blood and regulate extracellular ATP level via counteracting pyrophosphatase and phosphotransfer reactions. FASEB J 17:1328–1330

    CAS  Google Scholar 

  18. Lorenz WW, McCann RO, Longiaru M, Cormier MJ (1991) Isolation and expression of a cDNA encoding Renilla reniformis luciferase. Proc Natl Acad Sci USA 88:4438–4442

    Article  CAS  Google Scholar 

  19. Markova SV, Golz S, Frank LA, Kalthof B, Vysotski ES (2004) Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa. A novel secreted bioluminescent reporter enzyme. J Biol Chem 279:3212–3217

    Article  CAS  Google Scholar 

  20. Tannous BA, Kim DE, Fernandez JL, Weissleder R, Breakefield XO (2005) Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo. Mol Ther 11:435–443

    Article  CAS  Google Scholar 

  21. Greer LF 3rd, Szalay AA (2002) Imaging of light emission from the expression of luciferases in living cells and organisms: a review. Luminescence 17:43–74

    Article  CAS  Google Scholar 

  22. Weissleder R (2001) A clearer vision for in vivo imaging. Nat Biotechnol 19:316–317

    Article  CAS  Google Scholar 

  23. Loening AM, Wu AM, Gambhir SS (2007) Red-shifted Renilla reniformis luciferase variants for imaging in living subjects. Nat Meth 4:641–643

    Article  CAS  Google Scholar 

  24. Inouye S, Shimomura O (1997) The use of Renilla luciferase, Oplophorus luciferase, and apoaequorin as bioluminescent reporter protein in the presence of coelenterazine analogues as substrate. Biochem Biophys Res Commun 233:349–353

    Article  CAS  Google Scholar 

  25. Kataeva I, Chang J, Xu H, Luan CH, Zhou J, Uversky VN, Lin D, Horanyi P, Liu ZJ, Ljungdahl LG, Rose J, Luo M, Wang BC (2005) Improving solubility of Shewanella oneidensis MR-1 and Clostridium thermocellum JW-20 proteins expressed into Esherichia coli. J Proteome Res 4:1942–1951

    Article  CAS  Google Scholar 

  26. Kishi Y, Tanino H, Goto T (1972) The structure confirmation of the light-emitting moiety of bioluminescent jellyfish. Tetrahedron Lett 13:2747

    Article  Google Scholar 

  27. Kakoi H, Inoue S (1980) A new synthesis of Watasenia prelucifein by cyclization of 2-amino-3-benzyl-5-(p-hydroxyphenyl)pyrazine with p-hydroxyphenylpyruvic acid. Chem Lett 9:299–300

    Google Scholar 

  28. Shimomura O, Musicki B, Kishi Y (1988) Semi-synthetic aequorin. An improved tool for the measurement of calcium ion concentration. Biochem J 251:405–410

    CAS  Google Scholar 

  29. Vysotski ES, Liu ZJ, Rose J, Wang BC, Lee J (2001) Preparation and X-ray crystallographic analysis of recombinant obelin crystals diffracting to beyond 1.1 Å. Acta Crystallogr D Biol Crystallogr 57:1919–1921

    Article  CAS  Google Scholar 

  30. Illarionov BA, Frank LA, Illarionova VA, Bondar VS, Vysotski ES, Blinks JR (2000) Recombinant obelin: cloning and expression of cDNA, purification and characterization as calcium indicator. Methods Enzymol 305:223–249

    Article  CAS  Google Scholar 

  31. Klabusay M, Blinks JR (1996) Some commonly overlooked properties of calcium buffer systems: a simple method for detecting and correcting stoichiometric imbalance in CaEGTA stock solutions. Cell Calcium 20:227–234

    Article  CAS  Google Scholar 

  32. McCapra F, Chang YC (1967) The chemiluminescence of a Cypridina luciferin analogue. Chem Commun 19:1011–1012

    Google Scholar 

  33. Goto T (1968) Chemistry of bioluminescence. Pure Appl Chem 17:421–441

    Article  CAS  Google Scholar 

  34. Markova SV, Vysotski ES, Blinks JR, Burakova LP, Wang BC, Lee J (2002) Obelin from the bioluminescent marine hydroid Obelia geniculata: cloning, expression, and comparison of some properties with those of other Ca2+-regulated photoproteins. Biochemistry 41:2227–2236

    Article  CAS  Google Scholar 

  35. Frank LA, Borisova VV, Markova SV, Malikova NP, Stepanyuk GA, Vysotski ES (2008) Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay. Anal Bioanal Chem 391:2891–2896

    Article  CAS  Google Scholar 

  36. Rowe L, Dikici E, Daunert S (2009) Engineering bioluminescent proteins: expanding their analytical potential. Anal Chem 81:8662–8668

    Article  CAS  Google Scholar 

  37. Chiesa A, Rapizzi E, Tosello V, Pinton P, de Virgilio M, Fogarty KE, Rizzuto R (2001) Recombinant aequorin and green fluorescent protein as valuable tools in the study of cell signalling. Biochem J 355:1–12

    Article  CAS  Google Scholar 

  38. Pozzan T, Mongillo M, Rudolf R (2003) The Theodore Bücher lecture. Investigating signal transduction with genetically encoded fluorescent probes. Eur J Biochem 270:2343–2352

    Article  CAS  Google Scholar 

  39. Michelini E, Cevenini L, Mezzanotte L, Coppa A, Roda A (2010) Cell-based assays: fuelling drug discovery. Anal Bioanal Chem. doi:10.1007/s00216-010-3933-z

    Google Scholar 

Download references

Acknowledgments

This work was supported by grant 09-04-12022 of the Russian Foundation for Basic Research, “Molecular and Cell Biology” program of Russian Academy of Sciences, by the SB RAS grant No.2, and by the SB RAS Lavrentiev grant for Young Scientists.

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Authors and Affiliations

  1. Photobiology laboratory, Institute of Biophysics Russian Academy of Sciences, Siberian Branch, Krasnoyarsk, 660036, Russia

    Galina A. Stepanyuk, Natalia P. Malikova, Svetlana V. Markova & Eugene S. Vysotski

  2. Promega Biosciences, LLC, San Luis Obispo, CA, 93401, USA

    James Unch

  3. Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA, 30602, USA

    John Lee

Authors
  1. Galina A. Stepanyuk
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  2. James Unch
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  3. Natalia P. Malikova
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  4. Svetlana V. Markova
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  5. John Lee
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  6. Eugene S. Vysotski
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Corresponding author

Correspondence to Eugene S. Vysotski.

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Stepanyuk, G.A., Unch, J., Malikova, N.P. et al. Coelenterazine-v ligated to Ca2+-triggered coelenterazine-binding protein is a stable and efficient substrate of the red-shifted mutant of Renilla muelleri luciferase. Anal Bioanal Chem 398, 1809–1817 (2010). https://doi.org/10.1007/s00216-010-4106-9

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  • DOI: https://doi.org/10.1007/s00216-010-4106-9

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