Renilla GFP

The spectra of the luminescence of coelenterazine catalyzed by recombinant Renilla luciferase in the presence and absence of Renilla GFP are shown in Fig. 4.6.3 (Lorenz et al., 1991). Note that the luminescence intensity at the emission peak is increased more than... 

Fig. 4.6.3 Bioluminescence emission spectra measured with coelenterazine plus 1 i.M Renilla luciferase in the absence (a) and presence (b) of 1 jlM Renilla GFP. From Lorenz et al., 1991.

Table 4.7.1 A Comparison of Aequorea GFP and Renilla GFP (Ward, 1998, modified)...

Until 1999 Renilla GFP was the only biochemically well characterized fluorescent protein besides the Aequorea GFP. It proved to have a much higher extinction coefficient, resistance to pH-induced conformational changes and denaturation, and tendency to dimerize compared to Aequorea GFP [7]. However, chromophores from Aequorea and Renilla GFPs was shown to be chemically identical [7-9], indicating that the fluorescence properties of the proteins are depending on both, the chromophore and the corresponding environment provided by the amino-acid backbone. 

Until 1999 the Aequorea GFP remained the only cloned gene encoding a fluorescent protein. All the different GFP isoforms developed and used in molecular biology are derivatives of the GFP 10 cDNA cloned by Prasher and co-workers. Attempts to clone the gene for Renilla-GFP, which is supposed to be several times brighter than the Aequorea-GFP, were not successful. 

It was found that the excitation and emission spectra of green fluorescent protein (GFP) and its mutants are strongly pH dependent in aqueous solutions and intracellular compartments in living cells [51]. Typically, GFP and its mutant derivatives are fully fluorescent at pH values ranging from about pH 6.5 to 10. Above pH 10 fluorescence strongly declines. At pH values of 4 and below GFP is non-fluorescent, Fig. (8). Renilla GFP (rrenGFP2) shows a quite similar behavior. 

Quantum yield of luciferin. Various values of quantum yield have been reported for coelenterazine in the luminescence reaction catalyzed by Renilla luciferase 0.055 (Matthews et al., 1977a), 0.07 (Hart, et al., 1979), and 0.10-0.11 (with a recombinant form Inouye and Shimomura, 1997). The quantum yield is significantly increased in the presence of Renilla green fluorescent protein (GFP) see below. 

Like FRET, bioluminescence resonance energy transfer (BRET) is based on non-radiative energy transfer between a donor and an acceptor. However, in BRET, the donor is a luminescent molecule, excited by the enzyme Renilla Luciferase (Rluc), and not a fluorescent molecule. The BRET acceptor can be a fluorescent protein like green fluorescent protein (GFP) or YFR If the enzyme and the fluorescent protein are in close proximity (d < 10 nm), an energy transfer will occur between the Rluc substrate (coelanterazine) and the fluorescent protein, leading to emission from the later. 

Figure 2 Imaging protein-protein interactions by FRET and BRET, (a) Diagram illustrating FRET between the donor CFP-fusion and the acceptor YFP-fusion. (b) Detection of protein-protein interaction between X and Y by BRET using the Renilla luciferase (Rluc) as a bioluminescent donor and GFP as a fluorescent acceptor. The substrate for Rluc is deep blue coelenterazine. Constructs not drawn to scale.

Later on similar color shifts were found in the related coelenterates Obelia (a hydroid) and Renilla (a sea pansy) and it was suggested that the in-vivo excitation mechanism for coelenterate GFPs is based on radiationless energy transfer [3]. The first prove for this assumption was provided by Morise and coworkers in 1974 [4], They managed to purify and crystallize Aequorea GFP, and to demonstrate the efficient luminescence energy transfer between co-adsorbed Aequorin and GFP. In addition, the absorbance spectrum and fluorescence quantum yield was measured. 

Green fluorescent protein (GFP) is also a photoprotein isolated and cloned from the jellyfish Aequorea victoria. Variants have also been isolated from the sea pansy Renilla reniformis. GFP, like aequorin, produces a blue fluorescent signal, but without the required addition of an exogenous substrate. 

Wang Y, Wang G, O Kane DJ, Szalay AA. A study of protein protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP. Mol Gen Genet 2001 264 578-587. 

Ward, W.W. and Cormier, M.J., An energy transfer protein in coelenterate bioluminescence characterization of the Renilla green-fluorescent protein (GFP), /. Biol Chem., 254, 781-788,1979. 

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