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Green fluorescent protein

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File:1EMA GFP.png
GFP ribbon diagram. From PDB: 1EMA​.

The green fluorescent protein (GFP) is a protein, comprised of 238 amino acids (27 kDa), from the jellyfish Aequorea victoria that fluoresces green when exposed to blue light.[1][2] GFP has a unique can-like shape consisting of an 11-strand β-barrel with a single alpha helical strand containing the chromophore running through the center.[3][4] This barrel permits chromophore formation and protects it from quenching by the surrounding microenvironment. In cell and molecular biology, the GFP gene is frequently used as a reporter of expression.[5] 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 availability of GFP and its derivatives has thoroughly redefined fluorescence microscopy and the way it is used in cell biology and other biological disciplines.[6] 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).[7] 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.

History

In the 1960s and 70s GFP was first purified from A. victoria and its fluorescent properties studied by Osamu Shimomura.[8] In A. victoria, GFP fluorescence occurs when the luminescent protein 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.[9] However, its utility as a tool for molecular biologists was not realized until 1992 when Douglas Prasher reported the cloning and nucleotide sequence of GFP in Gene.[10] The funding for this project had run out, so Prasher sent cDNA samples to several labs. The lab of Martin Chalfie quickly expressed fluorescent GFP in heterologous cells of E. coli and C. elegans, publishing the results in Science in 1994.[11] Frederick Tsuji's lab independently reported the expression of the recombinant protein one month later.[12] Remarkably, the GFP molecule folded and was fluorescent at room temperature, without the need for exogenous cofactors specific to the jellyfish. Although this wild-type GFP 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.[3] One month later, the Phillips group independently reported the wild type GFP structure in Nature Biotech.[4] 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.

Mutagenesis

The diversity of genetic mutations is illustrated by this San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins.

Due to the potential for widespread usage and the evolving needs of researchers, many different mutants of GFP have been engineered.[13] The first major improvement was a single point mutation (S65T) reported in 1995 in Nature by Tsien.[14] 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). 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.[15]

Many other mutations have been made, including color mutants; in particular cyan fluorescent protein (ECFP, Cerulean, CyPet) and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet). 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 subsituted 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.[16]

The wild-type GFP (wtGFP) 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.

GFP in fine art

Julian Voss-Andreae's GFP-based sculpture Steel Jellyfish (2006). The image shows the stainless steel sculpture on display at Friday Harbor Laboratories on San Juan Island (Wash., USA), the place of GFP's discovery.

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"[17], created sculptures based on the structure of GFP, including the 5'6" (1.70 m) tall "Green Fluorescent Protein" (2004)[18] 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.

Notes

  • Research scientists from National Taiwan University's Department of Animal Science and Technology reported the production of three fluorescent pigs in early 2006.

References

  1. ^ Prendergast F, Mann K (1978). "Chemical and physical properties of aequorin and the green fluorescent protein isolated from Aequorea forskålea". Biochemistry. 17 (17): 3448–53. PMID 28749.
  2. ^ a b Tsien R (1998). "The green fluorescent protein" (PDF). Annu Rev Biochem. 67: 509–44. PMID 9759496.
  3. ^ a b Ormö M, Cubitt A, Kallio K, Gross L, Tsien R, Remington S (1996). "Crystal structure of the Aequorea victoria green fluorescent protein". Science. 273 (5280): 1392–5. PMID 8703075.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  4. ^ a b Yang F, Moss L, Phillips G (1996). "The molecular structure of green fluorescent protein". Nat Biotechnol. 14 (10): 1246–51. PMID 9631087.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  5. ^ Phillips G (2001). "Green fluorescent protein--a bright idea for the study of bacterial protein localization". FEMS Microbiol Lett. 204 (1): 9–18. PMID 11682170.
  6. ^ Yuste R (2005). "Fluorescence microscopy today". Nat Methods. 2 (12): 902–4. PMID 16299474.
  7. ^ Chudakov D, Lukyanov S, Lukyanov K (2005). "Fluorescent proteins as a toolkit for in vivo imaging". Trends Biotechnol. 23 (12): 605–13. PMID 16269193.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  8. ^ Shimomura O, Johnson F, Saiga Y (1962). "Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea". J Cell Comp Physiol. 59: 223–39. PMID 13911999.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  9. ^ Morise H, Shimomura O, Johnson F, Winant J (1974). "Intermolecular energy transfer in the bioluminescent system of Aequorea". Biochemistry. 13 (12): 2656–62. PMID 4151620.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  10. ^ Prasher D, Eckenrode V, Ward W, Prendergast F, Cormier M (1992). "Primary structure of the Aequorea victoria green-fluorescent protein". Gene. 111 (2): 229–33. PMID 1347277.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  11. ^ Chalfie M, Tu Y, Euskirchen G, Ward W, Prasher D (1994). "Green fluorescent protein as a marker for gene expression". Science. 263 (5148): 802–5. PMID 8303295.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  12. ^ Inouye S, Tsuji F (1994). "Aequorea green fluorescent protein. Expression of the gene and fluorescence characteristics of the recombinant protein". FEBS Lett. 341 (2–3): 277–80. PMID 8137953.
  13. ^ Shaner N, Steinbach P, Tsien R (2005). "A guide to choosing fluorescent proteins" (PDF). Nat Methods. 2 (12): 905–9. PMID 16299475.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  14. ^ Heim R, Cubitt A, Tsien R (1995). "Improved green fluorescence" (PDF). Nature. 373 (6516): 663–4. PMID 7854443.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  15. ^ Pédelacq J, Cabantous S, Tran T, Terwilliger T, Waldo G (2006). "Engineering and characterization of a superfolder green fluorescent protein". Nat Biotechnol. 24 (1): 79–88. PMID 16369541.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  16. ^ Miesenböck G, De Angelis D, Rothman J (1998). "Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins". Nature. 394 (6689): 192–5. PMID 9671304.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  17. ^ Voss-Andreae, J (2005). "Protein Sculptures: Life's Building Blocks Inspire Art". Leonardo. 38: 41–45.
  18. ^ Pawlak, Alexander (2005). "Inspirierende Proteine". Physik Journal. 4: 12.
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