The fluorescent Protein (FP)
GFP a green fluorescent protein was discovered in 1955 (Davenport et al. 1955), but till it was cloned in 1992 it had hardly been studied (Figure 1a) (Prasher et al. 1992; Chalfie et al. 1994). After the cloning of Wild-type GFP (wtGFP) gene from the jellyfish Aequorea victoria, the great potential of GFP in cell biological research was widely recognized. Predominantly because in numerous cases fusions could be made between GFP and a protein of interest, this without loosing the biological function of the protein of interest. Such fusions facilitated tracking of proteins in time and space, in the living cell. But also the GFP characteristics make it very suited to be used as a fluorescent protein tag. It is a rather small protein (21 kDa) and does not require cofactors to become fluorescent. It has a cylindrical b-can structure. Eleven strands of a b-sheet form an anti-parallel barrel with short helices forming lids on each end of this can. The chromophore is positioned inside the can, as part of a distorted a-helix, which runs along the axis of the can.
(a) The jellyfish Aequorea victoria containing wtGFP (Maniatis et al. 2000). (b) Schematic representation of green fluorescent protein, tripeptide fluorophore in red (Varian Australia Pty Ltd; Maniatis et al. 2000).
This barrel structure protects the fluorescent chromophore from loosing its fluorescent properties by solvents or detergents. It also makes it temperature insensitive up to 65ºC, protease insensitive and active between pH values of 5.5-12.0. So in short, GFP is a rather robust fluorescent probe. GFP also can be used to make fusion proteins by means of recombinant DNA technology, it has a great potential to be used as fluorescent tag to study the behavior of proteins in living cells. By doing so the mentioned disadvantages of in vitro labeling with a synthetic fluorescent group can be eliminated. The impact GFP made in research is illustrated by the 15000 studies published last decade, in which GFP itself is studied or used as reporter (NCBI). Nowadays GFP expands now from basic science to more industrial applications: For example GFP is used to track meat-fermenting lactobacilli in Japanese sausages (Gory et al. 2001), the spread of bacteria that consume diesel oil in soil (Dandie et al. 2001) and GFP is even used to make fluorescent plants or fish for decorative purposes (Gong et al. 2003; Choi et al. 2004). So GFP went from “zero to hero”. GFP mutants for heterologous biological systems: To obtain efficient translation in heterologous biological systems some alterations in codon usage and amino acids (aa) of GFP were needed. Such GFPs mutation optimization procedures allowed expression in many organisms like dictostelium, yeast, and animals.
Spectral GFP mutants were made, which allow tracking of several fusion proteins in one cell simultaneous. So in addition to species related optimization also GFPs with markedly modified fluorescence properties have been made (Heim et al. 1995; Haseloff et al. 1997; Davis et al. 1998). This resulted in GFPs with shifted emission spectra, such as Blue FPs, Cyan FPs, Yellow FPs and Red FPs. Beside these GFP derivatives new FPs with different spectral properties were discovered for example Discosoma sp. DsRED and Heteractis crispa HcRED, All together this resulted in a good collection of FPs with different excitation and emission spectra.
Normalized excitation (a) and emission (b) spectra of several fluorescent proteins. Enhanced Blue FP(EBFP), Cyan FP (ECFP), Green FP (EGFP), Yellow FP (EYFP), Red FPs (DsRed from Discosoma sp. and it monomeric RFP (mRFP1) derivative and HcRed1 from Heteractis crispa) (Tsien 1998; Matz et al. 1999; Baird et al. 2000; Campbell et al. 2002; Nagai et al. 2002; Rizzo et al. 2004; Shanner et al. 2004) (graphs are adapted from Miyawaki et al. (Miyawaki et al. 2003)).
As shown the excitation and emission spectra of the FPs are sufficiently different to select FP couples that can be tracked simultaneously. It turned out even to be possible to follow 4 FPs simultaneously (Kato et al. 2002; Hutter 2004). Additional spectral data off FPs and other fluorescent dyes can be found at Pubspectra.
Mutations in GFP not only resulted in spectral variants, but also in some cases the spectral properties of mutated GFPs became depended on environmental parameters like intracellular ion concentrations or redox potentials. Such mutated forms can be used as sensors and provide subcellular information about such parameters (Miesenbock et al. 1998; Baird et al. 1999; Jayaraman et al. 2000; Sujatha et al. 2000; Nagai et al. 2001; Hanson et al. 2004). In addition to these more or less coincidently obtained sensor GFPs, sensors also can be tailor made by inserting a specific protein domain into the cylindrical s-can structure of GFP (Nagai et al. 2000; Nagai et al. 2001). An example of such a domain is the calmoduline domain, which can bind the cellular compound, calcium (Ca2+). Upon calcium binding the calmoduline-GFP barrel structure is distorted upon which also the fluorescence emission is altered. However, more [Ca2+] sensitive sensors (referred to as Cameleons) were made based on FRET.