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

摘要

Green-fluorescent protein (GFP) is the origin of the green bioluminescencencolor exhibited by several marine hydrozoans and anthozoans. The mechanism is believed tonbe F€ orster resonance energy transfer (FRET) within a luciferaseu0002GFP or photoproteinu0002nGFP complex. As the effect is found in vitro atmicromolar concentrations, for FRET to occurnthis complex must have an affinity in the micromolar range. We present here a fluorescencendynamics investigation of the recombinant bioluminescence proteins from the jellyfish Clytiangregaria, the photoprotein clytin in its Can2þ-discharged formthat is highly fluorescent (λmax =n506 nm) and its GFP (cgreGFP; λmax = 500 nm). Can2þ-discharged clytin shows anpredominant fluorescence lifetime of 5.7 ns, which is assigned to the final emitting state of the bioluminescence reaction product,ncoelenteramide anion, and a fluorescence anisotropy decay or rotational correlation time of 12 ns (20 u0002C), consistent with tightnbinding and rotation with the whole protein. A 34 ns correlation time combined with a translational diffusion constant andmolecularnbrightness from fluorescence fluctuation spectroscopy all confirm that cgreGFP is an obligate dimer down to nanomolarnconcentrations. Within the dimer, the two chromophores have a coupled excited-state transition yielding fluorescence depolariza-ntion 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-foldnexcess of Can2þ-discharged clytin, indicating no stable complexation below 0.2 mM. It is proposed that any bioluminescence FRETncomplex with micromolar affinity must be one formed transiently by the cgreGFP dimer with a short-lived (millisecond)nintermediate in the clytin reaction pathway.