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Aequorin is a photoprotein isolated from luminescent jellyfish (like various Aequorea species, e.g., Aequorea victoria) and a variety of other marine organisms. It was originally isolated from the coelenterate by Osamu Shimomura.
Mechanism of Action
The two components of aequorin reconstitute spontaneously, forming the functional protein. The protein bears three EF hand motifs that function as binding sites for Ca2+ ions. When Ca2+ occupies such sites, 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 aequorin: 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: It has a low leakage rate from cells, lacks phenomena of intracellular compartmentalization or sequestration, 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.
- Shimomura O (1995). "A short story of aequorin.". Biol Bull. (Biological Bulletin, Vol. 189, No. 1) 189 (1): 1–5. doi:10.2307/1542194. JSTOR 1542194. PMID 7654844.
- Shimomura O, Johnson FH, Saiga Y (1962). "Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea". J Cell Comp Physiol 59 (3): 223–239. doi:10.1002/jcp.1030590302. PMID 13911999.
- Blinks JR, Wier WG, Hess P, Prendergast FG (1982). "Measurement of Ca2+ concentrations in living cells". Prog Biophys Mol Biol 40 (1–2): 1–114. doi:10.1016/0079-6107(82)90011-6. PMID 6758036.
- Montero M, Brini M, Marsault R, Alvarez J, Sitia R, Pozzan T, Rizzuto R (1995). "Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells". EMBO J 14 (22): 5467–75. PMC 394660. PMID 8521803.
- Kendall JM, Badminton MN, Sala-Newby GB, Campbell AK, Rembold CM (1996). "Recombinant apoaequorin acting as a pseudo-luciferase reports micromolar changes in the endoplasmic reticulum free Ca2+ of intact cells". Biochem J 318: 383–7. PMC 1217633. PMID 8809023.
- Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikurak M, Tsien RY (1997). "Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin". Nature 388 (6645): 882–7. doi:10.1038/42264. PMID 9278050.
- Heim N, Griesbeck O (2004). "Genetically encoded indicators of cellular calcium dynamics based on troponin C and green fluorescent protein". J Biol Chem 279 (14): 14280–6. doi:10.1074/jbc.M312751200. PMID 14742421.