Early history,discovery, and expression of Aequorea green fluorescent protein,with a note on an unfinished experiment

The bioluminescent hydromedusan jellyfish, Aequorea victoria, emits a greenish light (λ_max = 508 nm) when stimulated electrically or mechanically. The light comes from photocytes located along the margin of its umbrella. The greenish light depends on two intracellular proteins working in consort: aequorin (21.4 kDa) and a green fluorescent protein (27 kDa). An excited state green fluorescent protein molecule results, which, on returning to the ground state, emits a greenish light. Similarly, a green light emission may be induced in the green fluorescent protein by exposing it to ultraviolet or blue light. Because the green light can be readily detected under a fluorescence microscope, the green fluorescent protein, tagged to a protein of interest, has been used widely as a marker to locate proteins in cells and to monitoring gene expression. This article reviews the work that took place leading to the discovery, cloning, and expression of the green fluorescent protein, with a note on an unfinished experiment. Microsc. Res. Tech. 73:785–796, 2010. © 2010 Wiley‐Liss, Inc.     

Oxygen Activation of Apo‐obelin–Coelenterazine Complex

Ca²⁺‐regulated photoproteins use a noncovalently bound 2‐hydroperoxycoelenterazine ligand to emit light in response to Ca²⁺ binding. To better understand the mechanism of formation of active photoprotein from apoprotein, coelenterazine and molecular oxygen, we investigated the spectral properties of the anaerobic apo‐obelin–coelenterazine complex and the kinetics of its conversion into active photoprotein after exposure to air. Our studies suggest that coelenterazine bound within the anaerobic complex might be a mixture of N7‐protonated and C2(?) anionic forms, and that oxygen shifts the equilibrium in favor of the C2(?) anion as a result of peroxy anion formation. Proton removal from N7 and further protonation of peroxy anion and the resulting formation of 2‐hydroperoxycoelenterazine in obelin might occur with the assistance of His175. It is proposed that this conserved His residue might play a key role both in formation of active photoprotein and in Ca²⁺‐triggering of the bioluminescence reaction.