The Green Fluorescent Protein (GFP) has been widely used in the determination of gene expression and localisation of expressed proteins in living organisms, and has thus revolutionised the field of biological sciences research. The chromophore, composed of just three amino acids, imparts green fluorescence to the biomolecule. Mutations in GFP have reduced the limitations of the wild type (wt) protein and made it more applicable. The remarkable properties of GFP are summarised in this review which have been exploited for a wide variety of applications.
Structural and Biochemical Properties
The GFP has become a very useful tool in recent years in many scientific fields such as molecular biology, cell biology and biotechnology. GFP was first discovered by Shimomura et al from the jellyfish Aequora victoria following the discovery of the luminescent protein Aequorin. But the protein began to gain much attention only after it was first cloned in 1992 by Prasher et al.
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The structural, absorbance and fluorescence properties of the originally discovered GFP are discussed herewith. GFP is a small protein consisting of 238 amino acids with a major emission peak at 395nm and a minor one at 475nm. In the physiological state, excitation at 395nm gives an emission at 508nm while excitation at 475nm gives an emission peaking at 503nm, both resulting in a green fluorescence. The minor excitation peak of GFP is close to the blue luminescence of aequorin and therefore the green fluorescence of GFP occurs by conversion of the blue glow of aequorin to a green glow, as a consequence of the ability of aequorin to be able to efficiently transfer its luminescent energy to GFP. The chromophore of the GFP, formed spontaneously by the residues Serine-Tyrosine-Glycine (65-66-67), is the part of the protein that is responsible for the green colour. Chemically, it is a 4-(p-hydroxybenzylidene) imidazolinone and it is attached to the peptide backbone of the protein. The gene for GFP has also been successfully expressed into various other organisms including plants, microbes and mammals, to generate fluorescence. This implies that the gene might not be requiring any enzymes/co-factors that would restrict its fluorescence to the jellyfish itself.
Aequorin Ca2+ Blue Fluorescent Protein + hνλ470nm + CO2 + GFP hνλ508nm
Schematic representation of the emission of green light from the Green Fluorescent Protein
The crystal structure of the GFP has revealed that it comprises of 11 β-strands which accommodate a hollow cylinder that consists of an α-helix attached to the chromophore. The cylinder has also been referred to as the β-can and has a diameter of 2.4nm and a height of 4.2nm and almost all the primary sequences are involved in the formation of the β-barrel and the α-helix. GFP has been found to be a monomer as well as a dimer upon crystallisation but this does not seem to be an inherent feature of GFP and thus this variation may depend upon the crystallisation procedures.
GFP can fold effectively at room temperature or below, but loses this efficiency greatly as the temperature increases. It is sensitive to temperature only till the folding process. Once it matures completely at a low temperature, it then remains stable and is able to fluoresce upto 65°C. But it is difficult for the GFP to mature at such high temperatures. This suggests that wt GFP is thermosensitive. Several techniques have been used to mutate the wt GFP in order to produce mutants with enhanced properties such as improved folding at high temperatures and increased ability of the protein to diffuse into target cells.
GFP has been employed in several molecular and cell biology applications where it has been used as a fluorescent fusion tag by attaching to a target protein and then detecting it by irradiating the cell with ultraviolet light. It has also become extremely popular as a fluorescent marker for gene expression by linking it to DNA promoters of interest. On reviewing several scientific papers, it was found that the GFP has been used in numerous applications and the properties of the wt-GFP have been improved by means of mutations(random/site directed) in the GFP gene and this has resulted in GFP's with enhanced brightness as well as GFP's with a wide variety of colours. As a result of the increased brightness, the mutants maybe required in much lower amounts, thus minimising their interference in natural cellular process, although it appears that they are relatively non-toxic and therefore do not disrupt the functions of proteins to which they are tagged. A relevant example to report here would be the single point mutation in which Serine 65 residue of the wt-GFP is altered into a Threonine residue (S65T), resulting in the development of one of the most sought after variant of the GFP, termed as the Enhanced Green Fluorescent Protein (EGFP).
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GFP's ability to serve as a reporter gene has been utilised to study the survival of mesenchymal stem cells from rat bone marrow after their transplantation into the rat brain, which indicates that its applicability is widening exponentially. A very recent and fascinating study reported the use of a GFP expressing bacterial strain (S.aureus) to study the susceptibility of hernia repair meshes to the formation of bacterial biofilm formation, which could revolutionise the choice of meshes in hernia repair and minimize complications.
It can be concluded that GFP serves as an indispensable experimental tool. Majority of the research is now focused towards development of the wt GFP with enhanced properties and characteristics so as to result in an improved visualisation ability of the cellular organisation and processes. It is apparent on reviewing the published literature that GFP and its variants will continue to be utilised in future scientific studies to advance our knowledge of the extraordinary applications this intriguing protein is capable of.