Spectral tuning of photoproteins by partnering site-directed mutagenesis strategies with the incorporation of chromophore analogs

L. Rowe, A. Rothert, C. Logue, C. M. Ensor, Sapna K Deo, Sylvia Daunert

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Aequorin and obelin are photoproteins whose calcium controlled bioluminescent light emission is used for labeling in assays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. Both of these photoproteins emit blue light from a 2- hydroperoxycoelenterazine chromophore, which is non-covalently bound in the hydrophobic core of the proteins. In an effort to produce aequorin and obelin variants with improved analytical properties, such as alternative emission colors and altered decay kinetics, seven mutants of aequorin and obelin were prepared and combined with 10 different coelenterazine analogs. These semi-synthetic photoprotein mutants exhibited shifts in bioluminescent properties when compared with wild-type proteins. The bioluminescent parameters determined for these semi-synthetic photoprotein mutants included specific activity, emission spectra and decay half-life time. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants that had significantly altered bioluminescent properties. The largest emission maxima shift obtained was 44 nm, and the largest decay half-life difference was 23.91 s.

Original languageEnglish
Pages (from-to)73-81
Number of pages9
JournalProtein Engineering, Design and Selection
Volume21
Issue number2
DOIs
StatePublished - Feb 1 2008
Externally publishedYes

Fingerprint

Luminescent Proteins
Mutagenesis
Chromophores
Site-Directed Mutagenesis
Aequorin
Calcium
Tuning
Proteins
Light emission
Labeling
Assays
Half-Life
Color
Imaging techniques
Kinetics
Light
obelin

Keywords

  • Aequorin
  • Bioluminescence
  • Cysteines
  • Obelin
  • Photoproteins

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology

Cite this

Spectral tuning of photoproteins by partnering site-directed mutagenesis strategies with the incorporation of chromophore analogs. / Rowe, L.; Rothert, A.; Logue, C.; Ensor, C. M.; Deo, Sapna K; Daunert, Sylvia.

In: Protein Engineering, Design and Selection, Vol. 21, No. 2, 01.02.2008, p. 73-81.

Research output: Contribution to journalArticle

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N2 - Aequorin and obelin are photoproteins whose calcium controlled bioluminescent light emission is used for labeling in assays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. Both of these photoproteins emit blue light from a 2- hydroperoxycoelenterazine chromophore, which is non-covalently bound in the hydrophobic core of the proteins. In an effort to produce aequorin and obelin variants with improved analytical properties, such as alternative emission colors and altered decay kinetics, seven mutants of aequorin and obelin were prepared and combined with 10 different coelenterazine analogs. These semi-synthetic photoprotein mutants exhibited shifts in bioluminescent properties when compared with wild-type proteins. The bioluminescent parameters determined for these semi-synthetic photoprotein mutants included specific activity, emission spectra and decay half-life time. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants that had significantly altered bioluminescent properties. The largest emission maxima shift obtained was 44 nm, and the largest decay half-life difference was 23.91 s.

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