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PEDS Advance Access published online on January 23, 2009
Protein Engineering Design and Selection, doi:10.1093/protein/gzn083
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Aequorin variants with improved bioluminescence properties

E. Dikici1,3, X. Qu1, L. Rowe1, L. Millner1, C. Logue1, S.K. Deo2, M. Ensor1 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 46202-3274, USA

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

The photoprotein aequorin has been widely used as a bioluminescent label in immunoassays, for the determination of calcium concentrations in vivo, and as a reporter in cellular imaging. It is composed of apoaequorin (189 amino acid residues), the imidazopyrazine chromophore coelenterazine and molecular oxygen. The emission characteristics of aequorin can be changed by rational design of the protein to introduce mutations in its structure, as well as by substituting different coelenterazine analogues to yield semi-synthetic aequorins. Variants of aequorin were created by mutating residues His16, Met19, Tyr82, Trp86, Trp108, Phe113 and Tyr132. Forty-two aequorin mutants were prepared and combined with 10 different coelenterazine analogues in a search for proteins with different emission wavelengths, altered decay kinetics and improved stability. This spectral tuning strategy resulted in semi-synthetic photoprotein mutants with significantly altered bioluminescent properties.

Keywords: aequorin/bioluminescence/coelenterazine analogues/emission wavelength/mutagenesis

Received September 17, 2008; revised December 19, 2008; accepted December 19, 2008.


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