From the jellyfish Aequorea victoria, Green Fluorescent Protein is a fascinating protein that has found many applications as a tool in the biological sciences. It has the amazing property of generating highly visible green light when excited in the 400 to 500 nm range. This fluorescence doesn't come from any chemical cofactor such as a flavin or heme, but from an intrinsic fluorophore created by a novel cyclization reaction of the poypeptide backbone.

Discovery

Green fluorescent protein was discovered in the lab of Saiga back in 1962 in Aequorea jellyfish in addition to the blue chemiluminescent protein, aequorin. It was first just reported as a footnote in a study of aequorin:
a protein giving solutions that look slightly greenish in sunlight though only yellowish under tungsten lights, and exhibiting a very bright, greenish fluorescence in the ultraviolet of a Mineralite, has also been isolated from squeezates.
They soon after characterized its peak emission at 508 nm, which was the same wavelength that live Aequorea tissue peaked at. The emission of aequorin was found to peak at 470 nm, which excited one of the major absorption bands of GFP. Thus it is believed that the energy from excited aequorin is transferred to GFP through Förster Resonance Energy Transfer (FRET). Similar proteins were found in other coelenterates such as hydroids and sea pansies. The first biochemical characterization of molecular weight and other properties occured in the lab of Frank Prendergast. The Prendergast group also correctly identified the chromophore responsible for the green fluorescence.

Structure

GFP is approximately 200 amino acids, and forms a large beta-barrel fold. The fluorophore is formed by the cyclization of three amino acids - Ser 65, Tyr 66 and Gly 67. The cyclization results in formation of a p-hydroxybenzylideneimidazolinone. Cyclization requires interaction with oxygen. The fluorophore sits on an alpha-helix which is surrounded by the beta-barrel like a sword in a sheath. The barrel protects the fluorophore from water, which quenches the fluorescence. The structure is surprisingly intolerant to mutation and truncation, making protein engineering a challenge.

Engineering

A number of mutants have been developed of GFP which change various properties. Most of these involve residues in the fluorophore, or those amino acids which are in direct contact with it. Changing amino acids around the fluorophore in order to allow the tyrosine to be deprotonated (go from a phenol to a phenolate) cause the 395 absorbance peak to disappear, leaving only the 475 nm peak. Replacing Tyr 66 with a Tryptophan results in a yellower emission from the protein. Other mutations can also give proteins that emit in the blue or the red. Some labs have reported evidence of FRET between different mutants of GFP.

Other, larger scale engineering projects have included inserting metal binding sites and receptors into GFP. If successful, one then has a fluorescent sensor for a particular metal or compound. Fusing of GFP to antibodies allows fluorescent detection of antigens.

Cellular and Genetic Applications

One of GFP's greatest strengths is the lack of an external chromophore. GFP fluoresces by itself, not requiring any chemical cofactor. Because of this unique property, it is possible to clone and express GFP in bacteria or tissue culture, or even live organisms and still detect the fluorescence. GFP has been added to transgenic mice, resulting in mice that glow green under ultraviolet radiation. By fusing GFP to another gene, one can track expression of that gene in the cell using a microscope which is lit with ultraviolet light. If one has several mutants of GFP that emit different colors, one may tag several proteins simultaneously and study coexpression. Because of the ability to get GFP into cells without physical manipulation, it has become an important tool in cellular and developmental genetics. One can now buy kits with a GFP plasmid, making this type of investigation very routine.

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