Aequorin

• UniProt: P07164

Aequorin is a photoprotein from bioluminescent jellyfish of the genus Aequorea. The best studied is aequorin from Aequorea victoria. In aequorin is coelenterazine, a luciferin, tied her. After the addition of calcium ions, to emit photons in vitro with a wavelength of lambda max = 470 nm ( A. victoria), and therefore it is also referred to as the " blue fluorescent protein ". In the following, reference is made to the aequorin from A. victoria.

History

Aequorin was originally isolated in 1961 by Osamu Shimomura from Aequorea victoria. The structure of coelenterazine, the bound luciferin, 1974 elucidated. In 1990, the gene for aequorin was cloned from A. victoria.

Structure

Aequorin consists of the apoprotein apo-aequorin (189 amino acids, 22 kDa) and its prosthetic group coelenterazine, which with a molecular weight of 423 much smaller luciferin. Coelenterazine is bound via a peroxide bridge to the protein.

The apoprotein contains four helix - loop-helix motifs ( EF -hands ), of which can bind three calcium ions. The crystal structure of aequorin was determined and published in 2000 with a resolution of 2.3 Å.

Aequorin is one of the most studied calcium ion -binding photoprotein. It is also thought that it is the result of calcium -binding proteins such as calmodulin regular.

Biochemistry

Aequorin contains a bound by Peroxidbrücke Coelenterazinmolekül, the actual luciferin. When the three binding sites are occupied by calcium ions, the conformation of the protein changes so that an intramolecular reaction is triggered. Consequently, coelenterazine is converted to an unstable dioxetane. After decarboxylation of the anion of Coeleteramid produced in an electronically excited state. After relaxation to the ground state, a photon is released.

Aequorin can be regenerated by release of the bound calcium ions by molecular oxygen and a new coelenterazine molecule. However, so far 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 bioluminescent radiation-free via the Förster resonance energy transfer of green fluorescent protein (GFP).

Applications

Since the aequorin bioluminescence calcium ions required, it can be used as an intramolecular calcium sensor. Back in 1967, the intracellular calcium concentration, twitch muscle fibers was analyzed. 1985, the cDNA was cloned aequorin. This allows transformation of the apoaequorin gene in different organisms (bacteria, yeasts, plants and animal cells). For example, could the cytosolic calcium concentration after certain stimuli, for example in plants ( wind or cold shock ) or yeasts (pheromones ) are measured.

Through attach a special sequence apoaequorin transported into special organelles. This allows the measurement of calcium concentration, for example, mitochondria or the nucleus. Fluorescent calcium ion -binding Chelatorfarbstoffe (e.g. Fura -2), however, are not so senstitiv and are hardly brought to the desired organelle in selectively.

As a calcium sensor aequorin is also popular because it can be connected with antibodies or proteins and has a very good signal -to-noise ratio. In all cases, the luciferin, coelenterazine to be available as a substrate for aequorin, which as a hydrophobic molecule but easily can diffuse through the cell membrane.