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 CO<sub>2</sub>. 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: Cd<sup>2+</sup>, Ba<sup>2+</sup>, Mn<sup>2+</sup>, Sr<sup>2+</sup>, Mg<sup>2+</sup>, 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.
Luminescent proteins originally isolated from marine or terrestrial organisms have played a key role in the development of several biosensing systems. These proteins have been used in a variety of applications including, immunoassays, binding assays, cell-based sensing, high throughput screening, optical imaging, etc. Among the luminescent proteins isolated, the bioluminescent protein aequorin has been one of the proteins at the forefront in terms of its use in a vast number of biosensing systems. In our laboratory, we have employed aequorin as a label in the development of highly sensitive assays through chemical and genetic modifications from single step analysis of physiologically important molecules in biological fluids. An important aspect of optimizing these assays for clinical use involves understanding the stability of the various aequorin variants that are available. To this end we have designed several stability studies involving three important aequorin mutants, Mutant S, Mutant 5, and Mutant 53. The cysteine free aequorin, Mutant S, has been the most ubiquitously used aequorin variant in our laboratory because of its increased stability and activity as compared to native aequorin. Mutant 5 and Mutant 53 contain a single cyteine residue at position 5 and 53 in the protein, respectively. Because of the presence of a single cysteine residue, Mutant 5 and Mutant 53 both can be site-specifically conjugated. This site specific conjugation capability gives Mutant 5 and Mutant 53 an advantage over native aequorin when developing assays. Additional studies optimizing the expression, purification, and charging of aequorin Mutant S were also performed. A thorough understanding of the efficient expression, purification, and storage of these aequorin mutants will allow for the more practical utilization of these mutants in the development of future biosensing systems.