Green-Fluorescent Protein from the Bioluminescent Jellyfish Clytia gregaria Is an Obligate Dimer and Does Not Form a Stable Complex with the Ca2+-Discharged Photoprotein Clytin

• Published in: Biochemistry (Easton) Vol. 50; no. 20; pp. 4232 - 4241
• Main Authors: Malikova, Natalia P, Visser, Nina V, van Hoek, Arie, Skakun, Victor V, Vysotski, Eugene S, Lee, John, Visser, Antonie J. W. G
• Format: Journal Article
• Language: English
• Published: American Chemical Society 24.05.2011
• Subjects: bacterial luciferase; correlation spectroscopy; energy-transfer; excitation transfer; lumazine protein; photobacterium-leiognathi; polarized fluorescence; recombinant obelin; refractive-index; vibrio-fischeri y1
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi101671p

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 Ca2+-discharged form that is highly fluorescent (λmax = 506 nm) and its GFP (cgreGFP; λmax = 500 nm). Ca2+-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 Ca2+-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.