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Proceedings Paper

EF-hand Ca2+-binding bioluminescent proteins: effects of mutations and alternative divalent cations
Author(s): Laura Rowe; Mark Ensor; Sylvia Daunert
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Paper Abstract

Bioluminescent photoproteins, such as aequorin and obelin, are proteins that emit light upon binding calcium. Aequorin and obelin contain four EF-hand domains arranged into a globular structure. The loop region of these EF-hand domains binds calcium by coordinating it in a pentagonal bipyramidal structure with oxygen atoms. The binding of calcium to these EF-hands causes a slight conformational change in the protein, which leads to the oxidation of the internally sequestered chromophore, coelenterazine, producing coelenteramide and CO2. The excited coelenteramide then relaxes radiatively, emitting bioluminescence at 471 nm in aequorin or 491 nm in obelin. Although calcium is the traditional, and generally the most powerful, triggering ligand in this bioluminescence reaction, alternative di- and trivalent cations can also bind to the EF-hand loops and stimulate luminescence. Species capable of this cross-reactivity include: Cd2+, Ba2+, Mn2+, Sr2+, Mg2+, and several lanthanides. Magnesium is also known to modulate the bioluminescence of wild-type aequorin, increase its stability, and decrease its aggregation tendency. Both wild-type aequorin and wild-type obelin contain several cysteine residues, aequorin has three and obelin has five. It is believed that these cysteine residues play an important, but as of yet unknown, role in the bioluminescence of these proteins, since mutating most of these residues causes significant loss in bioluminescent activity. In order to explore whether or not these cysteine residues contributed to the specificity of the EF-hand domains for cations we generated four aequorin and obelin mutants and observed their luminescent intensity and decay kinetics by stimulation with calcium, barium, and magnesium. It was found that the cysteine mutations do appear to alter the effects that alternative divalent cations have on the bioluminescence of both aequorin and obelin.

Paper Details

Date Published: 14 February 2007
PDF: 8 pages
Proc. SPIE 6449, Genetically Engineered and Optical Probes for Biomedical Applications IV, 64490T (14 February 2007); doi: 10.1117/12.701170
Show Author Affiliations
Laura Rowe, Univ. of Kentucky (United States)
Mark Ensor, Univ. of Kentucky (United States)
Sylvia Daunert, Univ. of Kentucky (United States)

Published in SPIE Proceedings Vol. 6449:
Genetically Engineered and Optical Probes for Biomedical Applications IV
Samuel Achilefu; Alexander Pavlovich Savitsky; Rebekka M. Wachter; Darryl J. Bornhop; Ramesh Raghavachari, Editor(s)

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