Firefly luciferase: Alteration of the color of emitted light resulting from substitutions at position 286

Journal of the American Chemical Society

Volumn 118, Issue 30, 1996, Pages 7243-7244

  Mamaev, Sergey V ^a   Laikhter, Andrei L ^a   Arslan, Tuncer ^a   Hecht, Sidney M ^a  

^a UNIVERSITY OF VIRGINIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

LUCIFERASE;

AMINO ACID SEQUENCE; ARTICLE; ESCHERICHIA COLI; GENE EXPRESSION; INSECT; MUTATION; NONHUMAN; SPECTROPHOTOMETRY;

EID: 0029743320     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja961053c     Document Type: Article

References (38)

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4. Light emission is apparently due to an oxyluciferin dianion at pH > 6.7 that produces yellow-green light; protonation at lower pH affords a red emission band. See, e.g.: (a) White, E. H.; Steinmetz, M. G.; Miano, J. D.; Wildes, P. D.; Morland, R. J. Am. Chem. Soc. 1980, 102, 3199. (b) Rosendahl, M. S.; Leonard, N. J.; DeLuca, M. Photochem. Photobiol. 1982, 35, 857.
5. Light emission is apparently due to an oxyluciferin dianion at pH > 6.7 that produces yellow-green light; protonation at lower pH affords a red emission band. See, e.g.: (a) White, E. H.; Steinmetz, M. G.; Miano, J. D.; Wildes, P. D.; Morland, R. J. Am. Chem. Soc. 1980, 102, 3199. (b) Rosendahl, M. S.; Leonard, N. J.; DeLuca, M. Photochem. Photobiol. 1982, 35, 857.
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8. This luciferase is homologous with L, cruciata luciferase at four of the five sites capable of controlling the wavelength of light emitted by the latter species, including Ser286.
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23. Purchased from Pharmacia Biotech.
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26. For elaboration and assay of luciferase in E. coli. the cells harboring pTrcLuc or pTrcLuc-St286 were cultured overnight at 25 °C in 3 mL of Luria-Bertan broth containing 50μg/mL ampicillin and 1 mM IPTG. The cells were harvested by centrifugation. resuspended in 100 μL of lysis buffer (100 mM K phosphate. pH 7.8, containing 1 mM EDTA and 1 mg/mL lysozyme). incubated at 25 °C for 30 min, and then frozen in dry ice. The frozen pellets were allowed to thaw at 25 °C: following centrifugation 20 μL of the cleared lysate was added to 100 μL of a commercial luciferase assay solution (Promega), and the spectrum of emitted light was recorded at pH 7.8 on a fluorescence spectrophotometer.
27. No luciferase activity resulted when pTrcLuc-St286 was transformed into E. coli lacking a suppressor tRNA (cf. Table 1). consistent with the insertion of amino acid 286 from a suppressor tRNA.
28. 7a
29. In addition to the dependence of luciferase production on the presence of an aminoacylated suppressor tRNA (Table 2), three of the activated suppressor tRNAs (containing phenylalanine, valine, and glucosylserine (3)) were used for the elaboration of dihydrofolate reductase (DHFR) from a mRNA containing a UAG codon at position 10. The derived DHFRs, which formed only in the presence of the activated suppressor tRNAs, were degraded with Glu-C endopeptidase, affording a peptide encompassing amino acids 1-17 of DHFR. These peptides were shown to be identical with authentic standards, as judged by HPLC analysis in systems highly sensitive to alteration of polypeptide structure.
30. 8 the syntheses of the serine phosphorate and O-glucosylserine derivatives are provided as supporting information.
31. Normalization of light intensity for the amount of luciferase protein actually produced in each case revealed that the luciferases containing serine phosphonate and glucosylated serine produced light somewhat less efficiently than the wild type (Table 2). At present, it is not clear whether this is due to incorrect folding of some of the modified proteins, or to an intrinsic property such as lesser population of the excited state or (partial) decay of the oxyluciferin dianion through a dark reaction.
32. Consistent with this interpretation, the thermostability of wild-type luciferase (58% loss of light production at 37 °C vs 25 °C) was much greater than that of luciferase containing Val286 (95% loss of light production).
33. See, however, ref 7c for introduction of PhnSer into a short peptide.
34. See, e.g.: (a) Baenziger, J. U. FASEB J. 1994, 8, 1019. (b) Lasky, L. A. Anna. Rev. Biochem. 1995, 64, 113.
35. See, e.g.: (a) Baenziger, J. U. FASEB J. 1994, 8, 1019. (b) Lasky, L. A. Anna. Rev. Biochem. 1995, 64, 113.
36. See, e.g.: (a) Pawson, T. FASEB J. 1994, 8, 1113. (b) Crabtree, G. R.: Clipstone, N. A. Annu. Rev. Biochem. 1994, 63, 1045. (c) Hunter, T. Cell 1995, 80, 225.
37. See, e.g.: (a) Pawson, T. FASEB J. 1994, 8, 1113. (b) Crabtree, G. R.: Clipstone, N. A. Annu. Rev. Biochem. 1994, 63, 1045. (c) Hunter, T. Cell 1995, 80, 225.
38. See, e.g.: (a) Pawson, T. FASEB J. 1994, 8, 1113. (b) Crabtree, G. R.: Clipstone, N. A. Annu. Rev. Biochem. 1994, 63, 1045. (c) Hunter, T. Cell 1995, 80, 225.