Aequorin
| Aequorin-1 ( Aequorea victoria ) | ||
|---|---|---|
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| Ribbon model of aequorin-2 according to PDB 1EJ3 with highlighted tyrosine 184 (pink) and bound coelenterazine (blue) | ||
| Mass / length primary structure | 189 amino acids | |
| Cofactor | Ca 2+ | |
| Identifier | ||
| External IDs | ||
| Enzyme classification | ||
| EC, category | 1.13.12.5 , monooxygenase | |
| Substrate | Coelenterazine, luciferin + O 2 | |
| Occurrence | ||
| Parent taxon | Cnidaria | |
Aequorin is a photoprotein from bioluminescent jellyfish of the genus Aequorea . Aequorin from Aequorea victoria has been studied best . Coelenterazine , a luciferin , is bound in aequorin . After adding calcium ions, it emits light quanta with a wavelength of λ max = 470 nm ( A. victoria ) in vitro , which is why it is also known as the “blue fluorescent protein”. In the following, reference is made to the aequorin from A. victoria .
history
Aequorin was originally isolated from Aequorea victoria by Osamu Shimomura in 1961 . The structure of coelenterazine, the bound luciferin, was elucidated in 1974. In 1990 the gene for aequorin was cloned from A. victoria .
structure
Aequorin consists of the actual apoprotein apoaequorin (189 amino acids , 22 kDa ) and its prosthetic group coelenterazine , the much smaller luciferin with a molar mass of 423. Coelenterazine is bound to the protein via a peroxide bridge .
The apoprotein contains four helix-loop-helix motifs ( EF hands ), three of which can bind calcium ions. The crystal structure aequorin was determined in 2000 to a resolution of 2.3 Å and published.
Aequorin is one of the best studied calcium ion-binding photoproteins. It's also thought to have originated from regular calcium-binding proteins such as calmodulin .
biochemistry
Aequorin contains a coelenterazine molecule bound by a peroxide bridge, the actual luciferin. When the three binding sites are occupied with calcium ions, the conformation of the protein changes so that an intramolecular reaction is triggered. As a result, coelenterazine is converted into an unstable dioxetane . After decarboxylation , the anion of coeleteramide is formed in an electronically excited state . After relaxation to the ground state , a light quantum is released.
After the bound calcium ions have been released, Aequorin can be regenerated by molecular oxygen and a new molecule of coelenterazine. The exact mechanism is still unknown.
The quantum yield of this reaction is Q = 0.15-0.20.
In vivo activity
The jellyfish A. victoria glows blue-green, because aequorin transfers part of the energy of the bioluminescence reaction to the green fluorescent protein (GFP) via the Förster resonance energy transfer.
Applications
Since Aequorin's bioluminescence requires calcium ions, it can be used as an intramolecular calcium sensor. The intracellular calcium concentration of contracting muscle fibrils was analyzed as early as 1967 . In 1985 the aequorin cDNA was cloned. This permits the transformation of the gene for apoaequorin in different organisms ( bacteria , yeasts , plants and animal cells). For example, the cytosolic calcium concentration could be measured after certain stimuli, for example in plants (wind or cold shock) or yeasts ( pheromones ).
By attaching a special sequence, apoaequorin is transported into special organelles . This allows the calcium concentration to be measured e.g. B. in mitochondria or the nucleus . Fluorescent, calcium ion-binding chelator dyes (eg. B. Fura-2 ), however, are not as sensitive and selective so hardly be brought into the desired organelle.
Aequorin is also popular as a calcium sensor because it can be linked to antibodies or proteins and has a very good signal-to-noise ratio . In all cases, the luciferin coelenterazine must be present as a substrate for aequorin, which, as a hydrophobic molecule, can easily diffuse through the cell membrane .
literature
- Kendall, JM. and badminton, MN. (1998): Aequorea victoria bioluminescence moves into an exciting new era . In: Trends Biotechnol . 16 (5); 216-24; PMID 9621461
- Shimomura, O. (2005): The discovery of aequorin and green fluorescent protein . In: J Microsc. 217 (Pt 1); 1-15; PMID 15655058
Individual evidence
- ↑ Shimomura, O. et al. (1962): Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea . In: J Cell Comp Physiol . 59 ; 223-39 PMID 13911999
- ↑ Shimomura, O. et al. (1974): Mechanism of the luminescent intramolecular reaction of aequorin. In: Biochemistry 13 (16); 3278-3286; PMID 4152180
- ^ Campbell AK. et al. (1990): From Luc and Phot genes to the hospital bed . In: J Biolumin Chemilumin. 5 (2): 131-9; PMID 1970919
- ^ Head, JF. (2000): The crystal structure of the photoprotein aequorin at 2.3 A resolution . In: Nature 405 (6784): 372-6; PMID 10830969 ; PDF (free full text access)
- ↑ Tsuji FI. et al. (1995): Molecular evolution of the Ca (2 +) - binding photoproteins of the Hydrozoa . In: Photochem Photobiol. 62 (4); 657-61; PMID 7480150
- ↑ Kendall, JM & Badminton, MN (1998): Aequorea victoria bioluminescence moves into an exciting new era. In: Trends Biotechnol. 16 (5); 216-224. PMID 9621461
- ↑ Kendall JM, Badminton MN: Aequorea victoria bioluminescence moves into an exciting new era . In: Trends Biotechnol. . 16, No. 5, May 1998, pp. 216-24. PMID 9621461 .
- ^ Ridgway, EB. and Ashley, CC. (1967): Calcium transients in single muscle fibers . In: Biochem Biophys Res Commun . 29 (2); 229-34; PMID 4383681
- ↑ Inouye, S. et al. (1985): Cloning and sequence analysis of cDNA for the luminescent protein aequorin . In: Proc Natl Acad Sci USA 82 (10); 3154-8; PMID 3858813 ; PDF (free full text access)
- ↑ Knight, MR. et al. (1991): Transgenic plant aequorin reports the effects of touch and cold-shock and elicitors on cytoplasmic calcium . In: Nature 352 (6335); 524-526; PMID 1865907
- ↑ Nakajima-Shimada, J. et al. (1991): Monitoring of intracellular calcium in Saccharomyces cerevisiae with an apoaequorin cDNA expression system . In: Proc Natl Acad Sci USA 88 (15); 6878-82; PMID 1862111 ; PDF (free full text access)
- ↑ Rizzuto, R. et al. (1992): Rapid changes of mitochondrial Ca2 + revealed by specifically targeted recombinant aequorin . In: Nature 358 (6384); 325-7; PMID 1322496
- ↑ Brini, M. et al. (1993): Nuclear Ca2 + concentration measured with specifically targeted recombinant aequorin . In: EMBO J. 12 (12); 4813-9; PMID 8223490 ; PMC 413931 (free full text, PDF).