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PEDS Advance Access originally published online on January 5, 2008
Protein Engineering Design and Selection 2008 21(2):73-81; doi:10.1093/protein/gzm073
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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

L. Rowe1, A. Rothert1, C. Logue1, C.M. Ensor1, S.K. Deo2 and S. Daunert1,3

1Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA 2Department of Chemistry and Chemical Biology, Indiana University, Purdue University Indianapolis, Indianapolis, IN 46202, USA

3 To whom correspondence should be addressed. E-mail: daunert{at}uky.edu

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.

Keywords: aequorin/bioluminescence/cysteines/obelin/photoproteins

Received July 13, 2007; revised October 16, 2007; accepted November 14, 2007.


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E. Dikici, X. Qu, L. Rowe, L. Millner, C. Logue, S.K. Deo, M. Ensor, and S. Daunert
Aequorin variants with improved bioluminescence properties
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