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GFP ribbon diagram. From PDB 1EMA.

The green fluorescent protein (GFP) is a protein, comprised of 238 amino acids (23 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. Image File history File links Please see the file description page for further information. ... The title given to this article is incorrect due to technical limitations. ... Pembrey Airport is located in Pembrey Carmarthenshire, in Wales. ... Image File history File links Size of this preview: 600 × 600 pixels Full resolution (1600 × 1600 pixel, file size: 516 KB, MIME type: image/png)By Richard Wheeler (Zephyris) 2006. ... Image File history File links Size of this preview: 600 × 600 pixels Full resolution (1600 × 1600 pixel, file size: 516 KB, MIME type: image/png)By Richard Wheeler (Zephyris) 2006. ... The Protein Data Bank (PDB) is a repository for 3-D structural data of proteins and nucleic acids. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... In chemistry, an amino acid is any molecule that contains both amino and carboxylic acid functional groups. ... The unified atomic mass unit (u), or Dalton (Da), is a small unit of mass used to express atomic and molecular masses. ... Orders Stauromedusae Coronatae Semaeostomeae Rhizostomae Jellyfish are marine invertebrates belonging to the Scyphozoan class. ... Binomial name Aequorea victoria (Murbach and Shearer, 1902) Aequorea victoria is a luminescent jellyfish found off the west coast of North America. ... Fluorescence induced by exposure to ultraviolet light in vials containing various sized Cadmium selenide (CdSe) quantum dots. ... A chromophore is part (or moiety) of a molecule responsible for its color. ... Quenching refers to any process which decreases the fluorescence intensity of a given substance. ... Cell biology (also called cellular biology or formerly cytology, from the Greek kytos, container) is an academic discipline that studies cells. ... Molecular biology is the study of biology at a molecular level. ... For a non-technical introduction to the topic, see Introduction to Genetics. ... In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to another gene of interest in cell culture, animals or plants. ... A biosensor is a device for the detection of an analyte that combines a biological component with a physicochemical detector component. ... Microscopy is any technique for producing visible images of structures or details too small to otherwise be seen by the human eye. ... Fluorescein in dropper used for eye examination. ... A phototoxic substance is a chemical compound which becomes toxic only when exposed to light. ... Wiktionary has a definition of: In vitro In vitro (Latin: within glass) means within a test tube, or, more generally, outside a living organism or cell. ... In vivo (Latin for (with)in the living). ... Viral vectors are a tool commonly used by biologists to deliver genetic material into cells inside a living organism or cultured in vitro. ...

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. Aequorin ribbon diagram from PDB database Aequorin is a photoprotein isolated from luminescent jellyfish (like various Aequorea species e. ... General Name, Symbol, Number calcium, Ca, 20 Chemical series alkaline earth metals Group, Period, Block 2, 4, s Appearance silvery white Standard atomic weight 40. ...

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] Image File history File linksMetadata Size of this preview: 600 × 600 pixels Full resolution (800 × 800 pixel, file size: 174 KB, MIME type: image/jpeg) A San Diego beach scene drawn with an eight color palette of bacterial colonies expressing fluorescent proteins derived from GFP and the red-fluorescent coral... Image File history File linksMetadata Size of this preview: 600 × 600 pixels Full resolution (800 × 800 pixel, file size: 174 KB, MIME type: image/jpeg) A San Diego beach scene drawn with an eight color palette of bacterial colonies expressing fluorescent proteins derived from GFP and the red-fluorescent coral... Fluorescein in dropper used for eye examination. ...

Many other mutations have been made, including color mutants; in particular blue fluorescent protein (EBFP, EBFP2, Azurite), cyan fluorescent protein (ECFP, Cerulean, CyPet) and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet). BFP derivatives 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 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. Yellow Fluorescent Protein (YFP) is a genetic mutant of green fluorescent protein, derived from Aequorea victoria. ... Fluorescent proteins localize the guanosine 5-triphosphate hydrolase ARF in the Golgi apparatus of a living macrophage. ...

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] Three different views of the high resolution structure of a truncated neuronal SNARE complex. ...

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. The wavelength is the distance between repeating units of a wave pattern. ... The visible spectrum (or sometimes optical spectrum) is the portion of the electromagnetic spectrum that is visible to (can be detected by) the human eye. ... Families Suborder Sessiliflorae Anthoptilidae Chunellidae Echinoptilidae Funiculinidae Kophobelemnidae Protoptilidae Renillidae Scleroptilidae Stachyptilidae Umbellulidae Veretillidae Suborder Subselliflorae Pennatulidae Pteroeididae Virgulariidae Sea Pens are colonial marine cnidarians belonging to the order Pennatulacea. ...

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]. Image File history File links Size of this preview: 800 × 514 pixels Full resolution (900 × 578 pixel, file size: 124 KB, MIME type: image/jpeg) Artist: Julian Voss-Andreae Sculpture shown: Steel Jellyfish, 2006. ... Image File history File links Size of this preview: 800 × 514 pixels Full resolution (900 × 578 pixel, file size: 124 KB, MIME type: image/jpeg) Artist: Julian Voss-Andreae Sculpture shown: Steel Jellyfish, 2006. ... Eduardo Kac was the first person to have a microchip implanted in his body. ...

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 [2].

Notes

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

Fluorescent green pigs were first bred by a group of researchers led by Prof. ...

References

External links

• Aglow in the Dark: Pieribone, V. and D.F. Gruber. 2006. The Revolutionary Science of Biofluorescence, Harvard University Press, 288p. (Popular science book describing history and discovery of GFP)
• Glowing Genes: A Revolution in Biotechnology, Prometheus Press - An easy to read book about GFP.
• Producing A GFP Fusion Protein
• Introduction to fluorescent proteins
• A fun and useful site about the history, uses and structure of GFP hosted by Marc Zimmer a chemist at Connecticut College
• Tsien Lab @ UCSD
• Excitation and emission spectra for various fluorescent proteins
• A paper about using GFP to study RNA dynamics
• Interactive Java applet demonstrating the chemistry behind the formation of the GFP fluorophore.
• Artist Julian Voss-Andreae's website