Green fluorescent protein

The green fluorescent protein (GFP) is a protein, comprised of 238 amino acids (26.9 kDa), originally isolated from the jellyfish Aequorea victoria/Aequorea aequorea/Aequorea forskalea that fluoresces green when exposed to blue light.^[1][2] The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm which is in the lower green portion of the visible spectrum. The GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm. In cell and molecular biology, the GFP gene is frequently used as a reporter of expression.^[3] In modified forms it has been used to make biosensors, and many animals have been created that express GFP as a proof-of-concept that a gene can be expressed throughout a given organism. The GFP gene can be introduced into organisms and maintained in their genome through breeding, or local injection with a viral vector can be used to introduce the gene. To date, many bacteria, yeast and other fungal cells, plant, fly, and mammalian cells have been created using GFP as a marker.

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Structure

GFP has a unique can-like shape consisting of an 11-strand β-barrel with a single alpha helical strand containing the fluorophore running through the center.^[4][5] While the tightly packed barrel shell protects the fluorophore from quenching by the surrounding microenvironment, the inward facing sidechains of the barrel induce specific cyclization reactions in the tripeptide Ser65–Tyr66–Gly67 that lead to fluorophore formation. This occurs in a series of discrete steps with distinct excitation and emission properties. This process is referred to as maturation.

History

Wild-type GFP (wtGFP)

In the 1960s and 70s GFP, along with the separate luminescent protein aequorin, was first purified from A. victoria and its properties studied by Osamu Shimomura.^[6] In A. victoria, GFP fluorescence occurs when aequorin interacts with Ca²⁺ ions, inducing a blue glow. Some of this luminescent energy is transferred to the GFP, shifting the overall color towards green.^[7] However, its utility as a tool for molecular biologists was not realized until 1992 when Douglas Prasher reported the cloning and nucleotide sequence of wtGFP in Gene.^[8] The funding for this project had run out, so Prasher sent cDNA samples to several labs. The lab of Martin Chalfie expressed the coding sequence of fluorescent GFP in heterologous cells of E. coli and C. elegans, publishing the results in Science in 1994.^[9] Frederick Tsuji's lab independently reported the expression of the recombinant protein one month later.^[10] Remarkably, the GFP molecule folded and was fluorescent at room temperature, without the need for exogenous cofactors specific to the jellyfish. Although this wtGFP was fluorescent, it had several drawbacks, including dual peaked excitation spectra, poor photostability and poor folding at 37°C.

The first reported crystal structure of a GFP was that of the S65T mutant by the Remington group in Science in 1996.^[4] One month later, the Phillips group independently reported the wild type GFP structure in Nature Biotech.^[5] These crystal structures provided vital background on chromophore formation and neighboring residue interactions. Researchers have modified these residues by directed and random mutagenesis to produce the wide variety of GFP derivaties in use today.

GFP derivatives

  Due to the potential for widespread usage and the evolving needs of researchers, many different mutants of GFP have been engineered.^[11] The first major improvement was a single point mutation (S65T) reported in 1995 in Nature by Roger Tsien.^[12] This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostablility and a shift of the major excitation peak to 488nm with the peak emission kept at 509 nm. This matched the spectral characteristics of commonly available FITC filter sets, increasing the practicality of use by the general researcher. The addition of the 37°C folding efficiency (F64L) point mutant to this scaffold yielded enhanced GFP (EGFP). EGFP has an extinction coefficient (denoted ε), also known as its optical cross section of 9.13×10^-21 m²/molecule, also quoted as 55,000 L/(mol·cm).^[13] Superfolder GFP, a series of mutations that allow GFP to rapidly fold and mature even when fused to poorly folding peptides, was reported in 2006.^[14]

Many other mutations have been made, including color mutants; in particular blue fluorescent protein (EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein (ECFP, Cerulean, CyPet) and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet). BFP derivatives (except mKalama1) contain the Y66H substitution. The critical mutation in cyan derivatives is the Y66W substitution, which causes the chromophore to form with an indole rather than phenol component. Several additional compensatory mutations in the surrounding barrel are required to restore brightness to this modified chromophore due to the increased bulk of the indole group. The red-shifted wavelength of the YFP derivatives is accomplished by the T203Y mutation and is due to π-electron stacking interactions between the substituted tyrosine residue and the chromophore.^[2] These two classes of spectral variants are often employed for fluorescence resonance energy transfer (FRET) experiments. Genetically-encoded FRET reporters sensitive to cell signaling molecules, such as calcium or glutamate, protein phosphorylation state, protein complementation, receptor dimerization and other processes provide highly specific optical readouts of cell activity in real time.

Semirational mutagenesis of a number of residues led to pH-sensitve mutants known as pHluorins, and later super-ecliptic pHluorins. By exploiting the rapid change in pH upon synaptic vesicle fusion, pHluorins tagged to synaptobrevin have been used to visualize synaptic activity in neurons.^[15]

The nomenclature of modified GFPs is often confusing due to overlapping mapping of several GFP versions onto a single name. For example, mGFP often refers to a GFP with an N-terminal palmitoylation that causes the GFP to bind to cell membranes. However, the same term is also used to refer to monomeric GFP, which is often achieved by the dimer interface breaking A206K mutation. Wild-type GFP has a weak dimerization tendency at concentrations above 5 mg/mL. mGFP also stands for "modified GFP" which has been optimized through amino acid exchange for stable expression in plant cells.

Utilization

The availability of GFP and its derivatives has thoroughly redefined fluorescence microscopy and the way it is used in cell biology and other biological disciplines.^[16] While most small fluorescent molecules such as FITC (fluorescein isothiocyanate) are strongly phototoxic when used in live cells, fluorescent proteins such as GFP are usually much less harmful when illuminated in living cells. This has triggered the development of highly automated live cell fluorescence microscopy systems which can be used to observe cells over time expressing one or more proteins tagged with fluorescent proteins. Analysis of such time lapse movies has redefined the understanding of many biological processes including protein folding, protein transport, and RNA dynamics, which in the past had been studied using fixed (i.e. dead) material.

Another powerful use of GFP is to express the protein in small sets of specific cells. This allows researchers to optically detect specific types of cells in vitro (in a dish), or even in vivo (in the living organism).^[17]

GFP in fine art

Alba, a fluorescent bunny, was commissioned by Eduardo Kac using GFP for purposes of art and social commentary [1].

Julian Voss-Andreae, a German-born artist specializing in "protein sculptures"^[18], created sculptures based on the structure of GFP, including the 5'6" (1.70 m) tall "Green Fluorescent Protein" (2004)^[19] and the 4'7" (1.40) tall "Steel Jellyfish" (2006). The latter sculpture is currently located at the place of GFP's discovery by Shimomura in 1962, the University of Washington's Friday Harbor Laboratories.Julian Voss-Andreae Sculpture. Retrieved on 2007-06-14.

Notes

Fluorescent green pigs were first bred by a group of researchers led by Wu Shinn-Chih at the Department of Animal Science and Technology at National Taiwan University, announcing the results of the experiment in January 2006.

The transgenic pigs were created by adding DNA encoding for the green fluorescent protein from fluorescent jellyfish to pig embryos which were then implanted in the utereus of female pigs. The pigs glow green in the dark, and have clearly green-tinged skin and eyes in daylight.

References

Further reading

• Pieribone V, Gruber D (2006). Aglow in the Dark: The Revolutionary Science of Biofluorescence. Cambridge: Belknap Press. ISBN 0674019210.  Popular science book describing history and discovery of GFP
• Zimmer M (2005). Glowing Genes: A Revolution In Biotechnology. Buffalo, NY: Prometheus Books. ISBN 1591022533. 

• Interactive Java applet demonstrating the chemistry behind the formation of the GFP fluorophore
• Tsien Lab @ UCSD
• Excitation and emission spectra for various fluorescent proteins
• The GFP Toolbox used for Drug Discovery