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. 1986 Nov;83(21):8107–8111. doi: 10.1073/pnas.83.21.8107

Site-specific mutagenesis of the calcium-binding photoprotein aequorin

Frederick I Tsuji *, Satoshi Inouye †,‡, Toshio Goto §, Yoshiyuki Sakaki †
PMCID: PMC386876  PMID: 16593774

Abstract

The luminescent protein aequorin from the jellyfish Aequoria victoria emits light by an intramolecular reaction in the presence of a trace amount of Ca2+. In order to understand the mechanism of the reaction, a study of structure-function relationships was undertaken with respect to modifying certain of its amino acid residues. This was done by carrying out oligonucleotide-directed site-specific mutagenesis of apoaequorin cDNA and expressing the mutagenized cDNA in Escherichia coli. Amino acid substitutions were made at the three Ca2+-binding sites, the three cysteines, and a histidine in one of the hydrophobic regions. Subsequent assay of the modified aequorin showed that the Ca2+-binding sites, the cysteines, and probably the histidine all play a role in the bioluminescence reaction of aequorin.

Keywords: bioluminescence, oxygenase, oligonucleotide-directed mutagenesis, protein modification

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Charbonneau H., Walsh K. A., McCann R. O., Prendergast F. G., Cormier M. J., Vanaman T. C. Amino acid sequence of the calcium-dependent photoprotein aequorin. Biochemistry. 1985 Nov 19;24(24):6762–6771. doi: 10.1021/bi00345a006. [DOI] [PubMed] [Google Scholar]
  2. Coffee C. J., Bradshaw R. A. Carp muscle calcium-binding protein. I. Characterization of the tryptic peptides and the complete amino acid sequence of component B. J Biol Chem. 1973 May 10;248(9):3305–3312. [PubMed] [Google Scholar]
  3. Collins J. H., Potter J. D., Horn M. J., Wilshire G., Jackman N. The amino acid sequence of rabbit skeletal muscle troponin C: gene replication and homology with calcium-binding proteins from carp and hake muscle. FEBS Lett. 1973 Nov 1;36(3):268–272. doi: 10.1016/0014-5793(73)80388-6. [DOI] [PubMed] [Google Scholar]
  4. Dedman J. R., Jackson R. L., Schreiber W. E., Means A. R. Sequence homology of the Ca2+-dependent regulator of cyclic nucleotide phosphodiesterase from rat testis with other Ca2+-binding proteins. J Biol Chem. 1978 Jan 25;253(2):343–346. [PubMed] [Google Scholar]
  5. Fullmer C. S., Wasserman R. H. The amino acid sequence of bovine intestinal calcium-binding protein. J Biol Chem. 1981 Jun 10;256(11):5669–5674. [PubMed] [Google Scholar]
  6. Goto T., Inoue S., Sugiura S. Cypridina bioluminescence. IV. Synthesis and chemiluminescence of 3,7-dihydroimidazo[1,2-a]pyrazin-3-one and its 2-methyl derivative. Tetrahedron Lett. 1968 Jul;(36):3873–3876. doi: 10.1016/s0040-4039(00)72353-7. [DOI] [PubMed] [Google Scholar]
  7. Hattori M., Sakaki Y. Dideoxy sequencing method using denatured plasmid templates. Anal Biochem. 1986 Feb 1;152(2):232–238. doi: 10.1016/0003-2697(86)90403-3. [DOI] [PubMed] [Google Scholar]
  8. Herzberg O., James M. N. Common structural framework of the two Ca2+/Mg2+ binding loops of troponin C and other Ca2+ binding proteins. Biochemistry. 1985 Sep 24;24(20):5298–5302. doi: 10.1021/bi00341a004. [DOI] [PubMed] [Google Scholar]
  9. Inouye S., Noguchi M., Sakaki Y., Takagi Y., Miyata T., Iwanaga S., Miyata T., Tsuji F. I. Cloning and sequence analysis of cDNA for the luminescent protein aequorin. Proc Natl Acad Sci U S A. 1985 May;82(10):3154–3158. doi: 10.1073/pnas.82.10.3154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kemple M. D., Ray B. D., Jarori G. K., Rao B. D., Prendergast F. G. Electron paramagnetic resonance of spin-labeled aequorin. Biochemistry. 1984 Sep 11;23(19):4383–4390. doi: 10.1021/bi00314a022. [DOI] [PubMed] [Google Scholar]
  11. Kobayashi T., Takasaki Y., Takagi T., Konishi K. The amino acid sequence of sarcoplasmic calcium-binding protein obtained from sandworm, Perinereis vancaurica tetradentata. Eur J Biochem. 1984 Oct 15;144(2):401–408. doi: 10.1111/j.1432-1033.1984.tb08478.x. [DOI] [PubMed] [Google Scholar]
  12. Kretsinger R. H., Nockolds C. E. Carp muscle calcium-binding protein. II. Structure determination and general description. J Biol Chem. 1973 May 10;248(9):3313–3326. [PubMed] [Google Scholar]
  13. Mitchell G. W., Hastings J. W. A stable, inexpensive, solid-state photomultiplier photometer. Anal Biochem. 1971 Jan;39(1):243–250. doi: 10.1016/0003-2697(71)90481-7. [DOI] [PubMed] [Google Scholar]
  14. Ray B. D., Ho S., Kemple M. D., Prendergast F. G., Nageswara Rao B. D. Proton NMR of aequorin. Structural changes concomitant with calcium-independent light emission. Biochemistry. 1985 Jul 30;24(16):4280–4287. doi: 10.1021/bi00337a005. [DOI] [PubMed] [Google Scholar]
  15. SHIMOMURA O., JOHNSON F. H., SAIGA Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol. 1962 Jun;59:223–239. doi: 10.1002/jcp.1030590302. [DOI] [PubMed] [Google Scholar]
  16. Shimomura O., Johnson F. H., Morise H. Mechanism of the luminescent intramolecular reaction of aequorin. Biochemistry. 1974 Jul 30;13(16):3278–3286. doi: 10.1021/bi00713a016. [DOI] [PubMed] [Google Scholar]
  17. Shimomura O., Johnson F. H. Peroxidized coelenterazine, the active group in the photoprotein aequorin. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2611–2615. doi: 10.1073/pnas.75.6.2611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Shimomura O., Johnson F. H. Regeneration of the photoprotein aequorin. Nature. 1975 Jul 17;256(5514):236–238. doi: 10.1038/256236a0. [DOI] [PubMed] [Google Scholar]
  19. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  20. Wallace R. B., Johnson M. J., Hirose T., Miyake T., Kawashima E. H., Itakura K. The use of synthetic oligonucleotides as hybridization probes. II. Hybridization of oligonucleotides of mixed sequence to rabbit beta-globin DNA. Nucleic Acids Res. 1981 Feb 25;9(4):879–894. doi: 10.1093/nar/9.4.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Watterson D. M., Sharief F., Vanaman T. C. The complete amino acid sequence of the Ca2+-dependent modulator protein (calmodulin) of bovine brain. J Biol Chem. 1980 Feb 10;255(3):962–975. [PubMed] [Google Scholar]
  22. Wilkinson J. M. The amino acid sequence of troponin C from chicken skeletal muscle. FEBS Lett. 1976 Nov;70(1):254–256. doi: 10.1016/0014-5793(76)80769-7. [DOI] [PubMed] [Google Scholar]
  23. Yazawa M., Yagi K. The amino acid sequence of the calmodulin obtained from sea anemone (metridium senile) muscle. Biochem Biophys Res Commun. 1980 Sep 16;96(1):377–381. doi: 10.1016/0006-291x(80)91225-5. [DOI] [PubMed] [Google Scholar]
  24. de Boer H. A., Comstock L. J., Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21–25. doi: 10.1073/pnas.80.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]

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