Concept of GFP – Green Fluorescent Protein
The Green Fluorescent Protein, GFP, is the first naturally fluorescent protein that has been identified. Its discovery has revolutionized the life sciences, allowing visualizing cells, organelles and biological processes, among others.
Structure and biochemical properties
GFP is a small protein composed of 238 amino acids that make up eleven antiparallel β chains, which together form a cylinder in the centre of which is an α helix. After cyclization and oxidation of three of the amino acids of the central helix, the amino acids serine, tyrosine and glycine (65, 66 and 67 positions ), a chromophore, a chemical group with ability to absorb and emit light, is formed. When an ultraviolet (UV) or blue light hits the chromophore, it absorbs the light and then releases energy emitting green light. The N-terminal and C-terminal ends of GFP are free and accessible to link with other proteins.
GFP is a stable protein, which tolerates temperatures up to 65° C and a pH of 5.5 to 12.2 and is resistant to prolonged treatments with proteases. It doesn’t require a substrate or cofactors for fluoresce.
The monomeric form of wild GFP presents a peak of excitement at 395nm and a smaller peak at 475nm, giving rise to a single emission at 509nm, located in the green area of the spectrum. The replacement of serine (Ser65) for threonine (Thr65) created a mutant and improved form of GFP, EGFP (Enhanced Green Fluorescent Protein), 6 times more fluorescent, which features a predominant peak at 488nm and a emission peak kept at 509nm.
GFP was discovered in 1962, by researcher Osamu Shimomura and his collaborators, at Friday Harbor laboratories, University of Washington, where they were studying the bioluminescence of jellyfish Aequorea victoria, a cnidaria present in the Pacific Ocean off the coast of North America. Shimomura isolated and purified a bioluminescent calcium-dependent protein, which he named aequorin (referring to the jellyfish) and verified the presence of another protein which featured a strong green fluorescence when exposed to UV light. This protein was then referred to as green fluorescent protein, or GFP.
They verified that when the aequorin binds to the calcium, it emits a blue light which is absorbed by GFP leading to the emission of a green light.
In the late 1980s, the American researcher Martin Chalfie began working with GFP in order to use the gene of this protein to view the activation of other genes and the subsequent production of proteins. Chalfie and his collaborators have identified the location of the gene responsible for synthesis of GFP in the genome of the Aequorea victoria. In 1994, they managed to incorporate this gene, through genetic manipulation, in the Escherichia coli bacterium, which display the typical GFP green light when illuminated with UV light. Next, the gene for GFP was inserted in the nematode Caenorhabdistis elegans and they were able to see and understand the development of nerve cells.
In 1996, another group of researchers eliminated the introns from GFP gene allowing its application in plants.
Later, Roger Y. Tsien amplified the chromatic spectrum of fluorescent proteins. The exchange of amino acids in the protein sequence of GFP allowed to obtain variants in the blue (BFP), cyan (CFP) and yellow (YFP) regions. Obtaining fluorescence in orange-red zone was only possible after the discovery of another protein GFP-like but with a red fluorescence, by Sergey Lukyanov. DsRED (Desired RED protein) was found in a bioluminescent coral of the genus Discosoma. Tsien and his collaborators have developed new variants of GFP to which they gave names of fruits according to the colour presented: mPlum, mCherry, mStrawberry, mOrange and mCitrine. Later, other researchers have contributed to the widening of the spectrum, allowing the marking with different colours and the observation of multiple processes simultaneously.
In 2008, Osamu Shimomura, Martin Chalfie and Roger Tsein have been awarded the Nobel Prize in Chemistry for the discovery and the development of studies and applications of GFP as a marker.
Applications of fluorescent proteins
Fluorescent proteins are very versatile and are used whether in microbiology or genetic engineering and Physiology. Through recombinant DNA techniques, GFP gene (or similar proteins) can be introduced in living cell cultures, or in specific cells present in an intact organism.
Fluorescent proteins are used as probes and, as such, allow the observation of processes until then invisible, such as the development of brain cells, the growth and spread of cancer cells, the development of Alzheimer’s disease, the growth of pathogenic bacteria, the spread of AIDS virus, the infection process of parasites (e.g. on Chagas disease), the evolution of the first cells of the embryo, among many others. They are also applied in the environmental biotechnology area for the detection of heavy metals and trinitrotoluene (TNT) in water wells or bore water. In this case, it was used genetically modified bacteria which are resistant to a pollutant and that fluoresce in its presence.
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