Green fluorescent protein has natural fluorescent properties which can be used in various applications. Mutagenesis of wild type GFP gives variants that are widely used as protein fusion tags, as biomarkers, used in studying protein interactions and localization of signals. GFP variants have led to technological advancement in biological studies such as FRET. In this review, we will study properties, uses, and variants of GFP developed in recent years.
The green fluorescent protein was first observed by Shimomura et al in Aequoria victoria. He suggested 4-p-hydroxybenzylidine imidazoline-5 as its chromophore, which is involved in the fluorescence activity of GFP (Tsien 1998). The expression of this gene was later explained by Chalfie et al (1994).
The chromophore which is made of 3 residues which are ser-tyr-gly (65-67) in the GFP protein makes the 4-p-hydroxybenzylidine imidazoline-5 structure later by post translation (Yang et al 1996).
The structure of GFP as suggested by Tsien is Î²-barrel with 11 stands. The chromophore lies in the centre of the Î²- barrel and is attached via alpha helix. The number of amino acids which make up the GFP is 238 (Tsien 1998).
PROPERTIES AND USES OF GFP
The wild type GFP from Aequoria victoria has an excitation spectrum of 395nm and 475nm (absorbs blue light) with an emission peak of 508nm and 503nm (maximum) (emit green light) respectively (Tsien 1988). The GFP can be excited by UV lamps as well as fluorescein isothiocynate (FITC) filters due to its spectral range (Margolin 2000).
The cloning and expression of GFP gene has given the important information of fluorophore synthesis in protein (Schwartz and Patterson 2006).The wild type GFP folds very slowly to attain active fluorescent state(mature chromophore) (Margolin 2000).The synthesis of fluorophore in GFP protein is an autocatalytic process. Mutagenesis of GFP tells that G67 is required for the fluorophore formation. The core fluorophore from Renilla reniformis is identical to A.victoria. It shows an excitation of 498nm (Yang et al 1996).
With the help of Mutagenesis variants of GFP were created, one of the point mutation was S65T which increased the speed of fluorophore formation. The other mutations Ala206-lys206, leu221-lys221, phe223-arg223 helped to overcome GFP dimerization (Schwartz and Patterson 2006).
There are 7 distinct classes of GFP variants based on chromophore component which acts on different absorbance and emission spectra (Tsien 1998).The Enhanced GFP (EGFP) variant has S65T mutation (improves fluorophore formation and brightness), phe64-leu64 (pacifies sensitivity to temperature), and codon optimization (expression in mammalian cells), which makes it a useful protein tag (Schwartz and Patterson 2006).
Figure 1. The above graph shows the absorbance and emission peaks of wtGFP and EGFP. The blue circles depict the major & minor absorbance peaks of wtGFP and green circles show the single emission peak of wtGFP. The blue and green squares show the absorbance and emission peak of EGFP respectively (Schwartz and Patterson 2006).
There is a progress in the development of cyan and yellow shifted mutants (CFP and YFP) from A. victoria, which are pH sensitive and mature faster than wild type (Chudakov et al 2005). Cerulean is a bright CFP developed by Rizzo et al to use it in FRET based sensors for glucokinase activation (2004).
GFP mutants can be used as fluorescent markers for time independent cell process. When mutants of GFPs are immobilized in aerated aqueous polymer gels and are excited at 488nm, they show repeated cycles of fluorescent emission (blinks several seconds). Hence, they are also used as molecular switches on optical storage elements (Dickson 1997).
Elowitz et al (1997) found that photoactivation of GFP takes place in presence of low oxygen. Among several photoactivatable proteins, PA-GFP (thr203-his203) from A. victoria was the first which have 100-fold increase green fluorescence at 517nm. KFP1 is a recently developed variant obtained from Anemonia sulcate which can be irradiated in reversible as well as irreversible ways upon green light irradiation (Chudakov 2005).
The plasmid vectors which are used to express proteins in bacteria use GFP fusion expression system. A number of proteins involved in cell division process in E. coli have been fused with GFP and expressed by lac promoter (Margolin 2000).
GFP fused with Dictostelium myosin cells was used to study the myosin activity. The expression of GFP myosin fused protein proved that myosin is involved in cytokinesis and development of Dictostelium discoideum ( Moores et al 1995)
GFP is protected from photobleaching by its rigid shell. Certain mutants are created by random combinations and directed mutagenesis (Kasprzak 2007). Major changes in fluorescence can be obtained by engineering the phosphorylation sites under defined conditions. FLIP (fluorescence loss in photobleaching) and FRAP (fluorescence recovery after photobleaching) are fluorescence imaging techniques are use to study protein kinetics, which is performed by photobleaching (Baker et al 2010). GFP along with these techniques is use to study gap junctions channels in living cells.
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FRET (fluorescence resonance energy transfer) is the most common technology used to create biochemically sensitive GFP variants. In this quantum mechanical phenomenon, the emission spectrums of two nearby fluorophores overlap the excitation spectrum of each other (one acts as a donor and the other as acceptor). It is also used to study the distance between protein residues and monitoring motor movements (actin or microtubules). The chromophores of GFP are labelled as donor and acceptor and are linked with motor proteins. There are 3 approaches namely, single pair molecule FRET (spFRET), Luminescence resonance energy transfer (LRET), and transient FRET measurements (Kasprzak 2007).
Figure 2. Image showing the use of GFP as fusion tag (middle), promoter activation (left) and protein interactions by using FRET (right). The florescent proteins used in FRET are shown by coloured barrels and target proteins as grey and black ovals (Chudakov 2005).
Miyawaki et al (1997) constructed indicators, which they called cameleons, for monitoring Ca+ signals in organelles and cytosol. The cameleons were created by using blue/cyan emitting GFP mutants, calmodulin, calmodulin binding peptide and blue/green emitting GFP. They used the FRET method.
Abad et al (2004) developed a chimera of GFP that is used as a probe for studying changes in mitochondrial matrix pH.
In summary, there are wide variants of GFP used in various applications. The variants allow multicolour labelling of cells for detection. It has given new perspective in fluorescent imaging techniques such as FRET, FLIP, and FRAP. Monitoring promoter activity and localization of signals have become simpler by the use of GFPs.