Green fluorescent protein (GFP) is a spontaneously fluorescent polypeptide of 27 kDa (238 amino acid residues) from the jellyfish Aequorea victoria that absorbs UV-blue light and emits in the green region of the spectrum (44). Its structure and potential uses for studying living cells have recently been reviewed in detail (45-50). The GFP chromophore results from a cyclization of three adjacent amino acids (S65, Y66, and G67) and the subsequent 1,2-dehydro-genation of the tyrosine (45-50). Although the chromophore by itself is able to absorb light, its fluorescent properties are associated with the presence of an 11-stranded P-barrel. GFP has been expressed in both bacteria and eukaryotic cells and has been produced by in vitro translation of the GFP mRNA. More
Eosin concentration (^iM)
Fig. 8. Influence of increasing eosin concentrations on fluorescence intensity of BSA trytophans (□) and on fluorescence intensity of BSA-bound eosin (■). Fluorescence intensities on the y-axis are in arbitrary units. BSA concentration was 1.8 |iM.
over, GFP retains its fluorescence when fused to heterologous proteins on the N- and C-terminals, and this binding generally does not affect the functionality of the tagged protein (45,46,49). This leads the way for the use of GFP as an intracellular reporter even if the fluorescence intensity of the native GFP is not bright enough for most intracellular applications. Variants have been engineered that have different excitation and/or emission spectra that better match available light sources (46,48-50). The use of brighter mutants was also found necessary in the case of low expression levels in specific cellular microenvironments (46-50). Some of them, such as the cyan (ECFP) and yellow (EYFP) fluorescent protein, have been specifically engineered to match the FRET criteria.
Although the use of GFP and GFP-variants chimera have given amazing information on protein synthesis, translocation, and intracellular localization, their structure limits their use for monitoring conformational changes in proteins. Because of their own size, GFP variants may modify the kinetics of con-formational changes when used for tagging small proteins. Furthermore, these variants are fused to the N- and/or C-terminals of the tagged protein, which is generally not the best location to be sensitive to conformational changes resulting from binding to other proteins or enzyme substrates. Thanks to recent progress in structural genomics, it has become easier to determine which kind of protein biological activities may change the distance between the N- and C-terminal tags above or below 10 nm (15).
Actually, GFP chimeras have demonstrated their potential for functional studies of proteins. This research field has been well covered by recent reviews (see refs. 28, 46, 47, 51, 52, and references therein). Intramolecular FRET was used to monitor protein activity especially for detecting UV-induced apoptosis and testing novel apoptosis inducers or inhibitors (53-57). All these studies were carried out with caspase fluorescent substrates that are labeled to show FRET when the substrate is intact. Caspase activity disrupts the substrate, inducing the disappearance of FRET. Unfortunately, this elegant method for monitoring enzyme activity is unable to give any information about potential associated conformational changes in the protein.
Phosphorylation is an efficient posttranscriptional way of modifying proteins to modulate protein functions. Different methods have been tested for monitoring phosphorylation through FRET methods. In special cases, phos-phorylation induces significant conformational changes that can be evidenced by FRET methods if the peptide can be tagged at both ends by GFP mutants. In this scheme, chimeric reporters have been constructed based on a double-tagged appropriate substrate peptide sequence for a given protein kinase (58,59). In this approach, a flexible linker sequence and a phosphorylation recognition domain that binds the phosphorylated substrate domain were included between two GFP variants. Thus, phosphorylation induces a huge conformational change that will change the distance and/or relative orientation between the GFP mutants. These changes will change the FRET conditions between the GFP mutants. In principle, this approach can be extended to any kind of kinases and the specific reporters can be expressed in both bacteria and mammalian cells (60,61). Nevertheless, although allowing efficient monitoring of the kinases' activity, this method does not give any information on the conformational changes experienced by kinases during the phosphorylation or dephos-phorylation processes.
Intermolecular FRET techniques have also been used for monitoring an interaction between a nuclear receptor and its specific activator that is required for transcription of transiently transfected and chromosomally integrated reporter genes (62). High-resolution FRET microscopy was used for the study of the potential role of "lipid rafts" in the interaction between cholera toxin P-subunits and GPI-anchored proteins in the plasma membrane of HeLa cells (63). The interaction of epidermal growth factor receptor and Grb2 in living cells has also been monitored using FRET imaging (64). In addition, in a study by Janetopoulos et al. (65), receptor-mediated activation of heterotrimeric G-proteins was visualized in living cells by monitoring FRET between a- and P-subunits fused to cyan and yellow fluorescent proteins (65). Their study deserves special mention because the protocol used allowed the determination of both the kinetics and the sites of G-protein activation in cells and tissues. For that purpose, the subunits Ga2 and GP of Dictyostelium discoideum were respectively tagged with cyan and yellow fluorescent proteins. FRET intensity was used to monitor changes in the G-protein concentration, reporting both protein activity and associated conformational changes.
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