|Aequorin ribbon diagram from PDB database with prosthetic group coelenterazine in blue|
Aequorin is a 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 will generate blue light.
Aequorin is composed of two distinct units, the apoprotein that is called apoaequorin and has an approximate molecular weight of 21 kDa, and the prosthetic group coelenterazine, the luciferin. This is to say, apoaequorin is an enzyme produced in the photocytes of the animal. 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 crystal structure revealed that aequorin binds coelenterazine and oxygen in the form of a peroxide, coelenterazine-2-hydroperoxide.
Mechanism of Action
Early studies of the bioluminescence of Aequorea by E. Newton Harvey had noted that the bioluminescence appears as a ring around bell, and occurs even in the absence of air. This was remarkable because most bioluminescence reactions appeared to require oxygen, and led to the idea that the animals somehow store oxygen. It was later discovered that the apoprotein can stably bind coelenterazine and oxygen is required for the regeneration to the active form of aequorin. However, in the presence of calcium ions, the protein undergoes a conformational change and through oxidation converts its prosthetic group, coelenterazine, into excited coelenteramide and CO2. As the excited coelenteramide relaxes to the ground state, blue light (wavelength = 469 nm) is emitted.
Uses in Biology and Medicine
Since the emitted light can be easily detected with a luminometer, aequorin has become a useful tool in molecular biology for the measurement of intracellular Ca2+ levels. Cultured cells expressing the aequorin gene can effectively synthesize apoaequorin: however, recombinant expression yields only the apoprotein, therefore it is necessary to add coelenterazine into the culture medium of the cells to obtain a functional protein and thus use its blue light emission to measure Ca2+ concentration. Coelenterazine is a hydrophobic molecule, and therefore is easily taken up across plant and fungal cell walls, as well as the plasma membrane of higher eukaryotes, making aequorin suitable as a (Ca2+ reporter) in plants, fungi, and mammalian cells.
Aequorin has a number of advantages over other Ca2+ indicators: because the protein is large, it has a low leakage rate from cells compared to lipophilic dyes such as DiI. It lacks phenomena of intracellular compartmentalization or sequestration as is often seen for Voltage-sensitive dyes, and does not disrupt cell functions or embryo development. Moreover the light emitted by the oxidation of coelenterazine does not depend on any optical excitation, so problems with auto-fluorescence are eliminated. The primary limitation of aequorin is that the prosthetic group coelenterazine is irreversibly consumed to produce light, and requires continuous addition of coelenterazine into the media. Such issues led to developments of other genetically encoded calcium sensors including the calmodulin-based sensor cameleon, developed by Roger Tsien and the troponin-based sensor, TN-XXL, developed by Oliver Griesbeck.
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