Site-specific mutagenesis of the calcium-binding photoprotein aequorin.
The luminescent protein aequorin from the jellyfish Aequoria victoria emits light by an intramolecular reaction in the presence of a trace amount of Ca(2+). In order to understand the mechanism of the reaction, a study of structure-function relationships was undertaken with respect to modifying certain of its amino acid residues. This was done by carrying out oligonucleotide-directed site-specific mutagenesis of apoaequorin cDNA and expressing the mutagenized cDNA in Escherichia coli. Amino acid substitutions were made at the three Ca(2+)-binding sites, the three cysteines, and a histidine in one of the hydrophobic regions. Subsequent assay of the modified aequorin showed that the Ca(2+)-binding sites, the cysteines, and probably the histidine all play a role in the bioluminescence reaction of aequorin.
Marine Biology Research Division, Scripps Institution of Oceanography, University of California-San Diego, La Jolla, CA 92093.
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