title: Green-fluorescent protein from the bioluminescent jellyfish Clytia gregaria is an obligate dimer and does not form a stable complex with the Ca(2+)-discharged photoprotein clytin.
abstract: Green-fluorescent protein (GFP) is the origin of the green bioluminescence color exhibited by several marine hydrozoans and anthozoans. The mechanism is believed to be Förster resonance energy transfer (FRET) within a luciferase-GFP or photoprotein-GFP complex. As the effect is found in vitro at micromolar concentrations, for FRET to occur this complex must have an affinity in the micromolar range. We present here a fluorescence dynamics investigation of the recombinant bioluminescence proteins from the jellyfish Clytia gregaria, the photoprotein clytin in its Ca(2+)-discharged form that is highly fluorescent (λ(max) = 506 nm) and its GFP (cgreGFP; λ(max) = 500 nm). Ca(2+)-discharged clytin shows a predominant fluorescence lifetime of 5.7 ns, which is assigned to the final emitting state of the bioluminescence reaction product, coelenteramide anion, and a fluorescence anisotropy decay or rotational correlation time of 12 ns (20 °C), consistent with tight binding and rotation with the whole protein. A 34 ns correlation time combined with a translational diffusion constant and molecular brightness from fluorescence fluctuation spectroscopy all confirm that cgreGFP is an obligate dimer down to nanomolar concentrations. Within the dimer, the two chromophores have a coupled excited-state transition yielding fluorescence depolarization via FRET with a transfer correlation time of 0.5 ns. The 34 ns time of cgreGFP showed no change upon addition of a 1000-fold excess of Ca(2+)-discharged clytin, indicating no stable complexation below 0.2 mM. It is proposed that any bioluminescence FRET complex with micromolar affinity must be one formed transiently by the cgreGFP dimer with a short-lived (millisecond) intermediate in the clytin reaction pathway.
authors.affiliation: Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences , Siberian Branch, Krasnoyarsk, Russia.
authors.forename: Natalia P
authors.initials: NP
authors.lastname: Malikova
authors.forename: Nina V
authors.initials: NV
authors.lastname: Visser
authors.forename: Arie
authors.initials: A
authors.lastname: van Hoek
authors.forename: Victor V
authors.initials: VV
authors.lastname: Skakun
authors.forename: Eugene S
authors.initials: ES
authors.lastname: Vysotski
authors.forename: John
authors.initials: J
authors.lastname: Lee
authors.forename: Antonie J W G
authors.initials: AJ
authors.lastname: Visser
mesh_terms: D000042:Absorption; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism*; D037402:Hydrozoa; D008164:Luminescent Proteins / Q000378:metabolism*; D008958:Models, Molecular; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D008164:Luminescent Proteins; C046165:phialidin; D049452:Green Fluorescent Proteins; D002118:Calcium