Aequorin

From Wikipedia, the free encyclopedia
Jump to: navigation, search
Aequorin 1
Aequorin 1EJ3.png
Aequorin ribbon diagram from PDB database with prosthetic group coelenterazine in blue
Identifiers
Organism Aequorea victoria (Jellyfish)
Symbol  ?
UniProt P07164
Other data
EC number 1.13.12.5

Aequorin is a photoprotein isolated from the hydrozoan Aequorea victoria.[1] Though the bioluminescence was studied decades before, the protein was originally isolated from the animal by Osamu Shimomura.[2] 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.

Discussions of "jellyfish DNA" to make "glowing" animals often refer to transgenic animals which express the Green fluorescent protein, not aequorin.

Structure[edit]

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.[3] 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.[4] The crystal structure revealed that aequorin binds coelenterazine and oxygen in the form of a peroxide, coelenterazine-2-hydroperoxide.[5]

Mechanism of Action[edit]

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.[6] This was remarkable because most bioluminescence reactions appeared to require oxygen, and led to the idea that the animals somehow store oxygen.[7] It was later discovered that the apoprotein can stably bind coelenterazine and oxygen is required for the regeneration to the active form of aequorin.[8] 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.[9] As the excited coelenteramide relaxes to the ground state, blue light (wavelength = 469 nm) is emitted.

Uses in Biology and Medicine[edit]

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.[10] 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.[11][12]

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.[13] 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,[14] developed by Roger Tsien and the troponin-based sensor, TN-XXL, developed by Oliver Griesbeck.[15]

References[edit]

  1. ^ 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. 
  2. ^ 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. 
  3. ^ Shimomura O, Johnson FH (1978). "Peroxidized coelenterazine, the active group in the photoprotein aequorin". PNAS USA 75 (3): 2611–2615. doi:10.1073/pnas.75.6.2611. PMC 392612. PMID 275832. 
  4. ^ Charbonneau H, Walsh KA, McCann RO, Prendergast FG, Cormier MJ, Vanaman TC (1985). "Amino acid sequence of the calcium-dependent photoprotein aequorin". Biochemistry 24 (24): 6762–71. doi:10.1021/bi00345a006. PMID 2866797. 
  5. ^ Head JF, Inouye S, Teranishi K, Shimomura O (2000). "The crystal structure of the photoprotein aequorin at 2.3 Å resolution". Nature 405 (6784): 372–6. doi:10.1038/35012659. PMID 10830969. 
  6. ^ Harvey EN (1926). "Oxygen and Luminescence, with a Description of Methods for Removing Oxygen from Cells and Fluids". Biological Bulletin 51 (2): 89–97. doi:10.2307/1536540. 
  7. ^ Harvey, E.N. "Bioluminescence" Academic Press., 1952.
  8. ^ Shimomura O, Johnson FH (1975). "Regeneration of the photoprotein aequorin". Nature 256 (5514): 236 – 238. doi:10.1038/256236a0. PMID 239351. 
  9. ^ Shimomura O, Johnson FH, Morise H (1974). "Mechanism of the luminescent intramolecular reaction of aequorin". Biochemistry 13 (16): 3278–3286. doi:10.1021/bi00713a016. PMID 4152180. 
  10. ^ Shimomura O, Inouye S, Musicki B, Kishi Y (1990). "Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators". Biochem. J. 270 (2): 309–12. PMC 1131721. PMID 2400391. 
  11. ^ 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. 
  12. ^ 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. 
  13. ^ 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. 
  14. ^ 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. 
  15. ^ 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. 

External links[edit]