Aequorin

Aequorin is a calcium-activated photoprotein isolated from the hydrozoan Aequorea victoria. Though the bioluminescence was studied decades before, the protein was originally isolated from the animal by Osamu Shimomura. In the animals, the protein occurs together with the green fluorescent protein to produce green light by resonant energy transfer, while aequorin by itself generates blue light. Aequorin is a calcium-activated photoprotein isolated from the hydrozoan Aequorea victoria. Though the bioluminescence was studied decades before, the protein was originally isolated from the animal by Osamu Shimomura. In the animals, the protein occurs together with the green fluorescent protein to produce green light by resonant energy transfer, while aequorin by itself generates blue light. Discussions of 'jellyfish DNA' to make 'glowing' animals often refer to transgenic animals which express the green fluorescent protein, not aequorin, although both originally derived from the same animal. Apoaequorin, a unit of aequorin, is an ingredient in the dietary supplement Prevagen. The US Federal Trade Commission (FTC) has charged the maker with false advertising for its memory improvement claims. Work on aequorin began with E. Newton Harvey in 1921. Though Harvey was unable to demonstrate a classical luciferase-luciferin reaction, he showed that water could produce light from dried photocytes and that light could be produced even in the absence of oxygen. Later, Osamu Shimomura began work into the bioluminescence of Aequorea in 1961. This involved tedious harvesting of tens of thousands of jellyfish from the docks in Friday Harbor, Washington. It was determined that light could be produced from extracts with seawater, and more specifically, with calcium. It was also noted during the extraction the animal creates green light due to the presence of the green fluorescent protein, which changes the native blue light of aequorin to green. While the main focus of his work was on the bioluminescence, Shimomura and two others, Martin Chalfie and Roger Tsien, were awarded the Nobel Prize in 2008 for their work on green fluorescent proteins. Aequorin is a holoprotein composed of two distinct units, the apoprotein that is called apoaequorin, which has an approximate molecular weight of 21 kDa, and the prosthetic group coelenterazine, the luciferin. This is to say, apoaequorin is the enzyme produced in the photocytes of the animal, and coelenterazine is the substrate whose oxidation the enzyme catalyzes. When coelenterazine is bound, it is called aequorin. Notably, the protein contains three EF hand motifs that function as binding sites for Ca2+ ions. The protein is a member of the superfamily of the calcium-binding proteins of which there are some 66 subfamilies. The crystal structure revealed that aequorin binds coelenterazine and oxygen in the form of a peroxide, coelenterazine-2-hydroperoxide. The binding site for the first two calcium atoms show a 20X greater affinity for calcium than the third site. However, earlier claims that only two EF-hands bind calcium, were questioned when later structures indicated that all three site indeed can bind calcium. Thus, titration studies show that all three calcium-binding sites are active but only two ions are needed to trigger the enzymatic reaction. Other studies have shown the presence of an internal cysteine bond that maintains the structure of aequorin. This has also explained the need for a thiol reagent like beta mercaptoethanol in the regeneration of the protein since such reagents weaken the sulfhydryl bonds between cysteine residues, expediting the regeneration of the aequorin. Chemical characterization of aequorin indicates the protein is somewhat resilient to harsh treatments. Aequorin is heat resistant. Held at 95⁰C for 2 minutes the protein lost only 25% activity. Denaturants 6M-urea or 4M-guanidine hydrochloride did not destroy the protein.