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[[Image:GFP structure.png|thumb|200px|Lu tiaggramma a nastru da prutiina fluoriscenti virdi (GFP, l'acronimu angrisi)]]
[[Image:Aequorea victoria.jpg|thumb|200px|''Aequorea victoria'']]
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Li sordi ppi stu pruggettu finièrru e allura Prasher mannau lu [[cDNA]] samples to several labs. The lab of [[Martin Chalfie]] expressed the coding sequence of wtGFP, with the first few amino acids deleted, in heterologous cells of ''[[E. coli]]'' and ''[[Caenorhabditis elegans|C. elegans]]'', publishing the results in ''Science'' in 1994.<ref name=Chalfie_1994>{{cite journal |author=Chalfie M, Tu Y, Euskirchen G, Ward W, Prasher D |title=Green fluorescent protein as a marker for gene expression |journal=Science |volume=263 |issue=5148 |pages=802–5 |year=1994 |pmid=8303295 |doi=10.1126/science.8303295}}</ref> Frederick Tsuji's lab independently reported the expression of the recombinant protein one month later.<ref name=Inouye_1994>{{cite journal |author=Inouye S, Tsuji F |title=Aequorea green fluorescent protein. Expression of the gene and fluorescence characteristics of the recombinant protein |journal=FEBS Lett |volume=341 |issue=2-3 |pages=277–80 |year=1994 |pmid=8137953 |doi=10.1016/0014-5793(94)80472-9}}</ref> Remarkably, the GFP molecule folded and was fluorescent at room temperature, without the need for exogenous cofactors specific to the jellyfish. Although this near-wtGFP was fluorescent, it had several drawbacks, including dual peaked excitation spectra, pH sensitivity, chloride sensitivity, poor fluorescence quantum yield, 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.<ref name="Ormo_1996"/> One month later, the Phillips group independently reported the wild type GFP structure in ''Nature Biotech''.<ref name="Yang_1996"/> 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 derivatives in use today. [[Martin Chalfie]], [[Osamu Shimomura]] and [[Roger Y. Tsien]] share the 2008 [[Nobel Prize in Chemistry]] for their discovery and development of the green fluorescent protein.<ref name='2008_Nobel'>{{cite news | first= | last= | coauthors= | title=The Nobel Prize in Chemistry 2008 | date=2008-10-08 | publisher= | url =http://nobelprize.org/nobel_prizes/chemistry/laureates/2008/ | work = | pages = | accessdate = 2008-10-08 | language = }}</ref>
 
===GFP derivatives===
[[Image:FPbeachTsien.jpg|thumb|200px|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.<ref name=Shaner_2005>{{cite journal |author=Shaner N, Steinbach P, Tsien R |title=A guide to choosing fluorescent proteins |journal=Nat Methods |volume=2 |issue=12 |pages=905–9 |year=2005 |url= http://tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Methods%20-%20Choosing%20fluorescent%20proteins.pdf |format=PDF|pmid=16299475 |doi=10.1038/nmeth819}}</ref> The first major improvement was a single point mutation (S65T) reported in 1995 in ''Nature'' by [[Roger Y. Tsien|Roger Tsien]].<ref name=Heim_1995>{{cite journal |author=Heim R, Cubitt A, Tsien R |title=Improved green fluorescence |journal=Nature |volume=373 |issue=6516 |pages=663–4 |year=1995 |url= http://tsienlab.ucsd.edu/Publications/Heim%201995%20Nature%20-%20Improved%20GFP.PDF |format=PDF|pmid=7854443 |doi=10.1038/373663b0}}</ref> This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostability and a shift of the major excitation peak to 488&nbsp;nm with the peak emission kept at 509&nbsp;nm. This matched the spectral characteristics of commonly available [[Fluorescein|FITC]] filter sets, increasing the practicality of use by the general researcher. A 37&nbsp;°C folding efficiency (F64L) point mutant to this scaffold yielding enhanced GFP (EGFP) was discovered in 1995 by the lab of Ole Thastrup.<ref name=Thastrup_1995>{{cite journal |author=Thastrup O, Tullin S, Kongsbak Poulsen L, Bjørn S |title = Fluorescent Proteins |year = 1995 |issue-date = 2001 |journal= US patent |patent-number = 6,172,188 |country-code = US |url=http://patft.uspto.gov/netacgi/nph-Parser?Sect2=PTO1&Sect2=HITOFF&p=1&u=%2Fnetahtml%2FPTO%2Fsearch-bool.html&r=1&f=G&l=50&d=PALL&RefSrch=yes&Query=PN%2F6172188}}</ref> EGFP opened for using GFPs in mammalian cells. EGFP has an extinction coefficient (denoted ε) of 55,000 M<sup>−1</sup>cm<sup>−1</sup>.<ref>{{cite journal | author=Shelley R. McRae, Christopher L. Brown and Gillian R. Bushell |title=Rapid purification of EGFP, EYFP, and ECFP with high yield and purity| journal=Protein Expression and Purification |volume=41|issue=1|month=May|year=2005|pages=121–127|url= |doi=10.1016/j.pep.2004.12.030 |pmid=15802229}}</ref> The fluorescence quantum yield (QY) of EGFP is 0.60. The relative brightness, expressed as ε•QY, is 33,000 M<sup>−1</sup>cm<sup>−1</sup>.
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.<ref name=Pedelacq_2006>{{cite journal |author=Pédelacq J, Cabantous S, Tran T, Terwilliger T, Waldo G |title=Engineering and characterization of a superfolder green fluorescent protein |journal=Nat Biotechnol |volume=24 |issue=1 |pages=79–88 |year=2006 |pmid=16369541 |doi=10.1038/nbt1172}}</ref>
 
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.<ref name="Tsien_1998"/> 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.
[[Image:Gfp and fluorophore.png|thumb|300px|GFP molecules drawn in cartoon style, one fully and one with the side of the [[beta barrel]] cut away to reveal the [[chromophore]] (highlighted as [[Ball-and-stick model|ball-and-stick]]). From {{PDB|1GFL}}.]]
Semirational mutagenesis of a number of residues led to pH-sensitive 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.<ref name=Miesenbock_1998>{{cite journal |author=Miesenböck G, De Angelis D, Rothman J |title=Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins |journal=Nature |volume=394 |issue=6689 |pages=192–5 |year=1998 |pmid=9671304 |doi=10.1038/28190}}</ref>
 
Redox sensitive versions of GFP ([[roGFP]]) were engineered by introduction of cysteines into the beta barrel structure. The [[redox]] state of the cysteines determines the [[fluorescent]] properties of [[roGFP]].<ref name=Hanson_2004>{{cite journal |author=Hanson GT, Aggeler R, Oglesbee D, Cannon M, Capaldi RA, Tsien RY, Remington SJ |title=Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators |journal=J Biol Chem |volume=279 |issue=13 |pages=13044–53 |year=2004 |pmid=14722062 |doi=10.1074/jbc.M312846200}}</ref>
 
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 membrane]]s. However, the same term is also used to refer to [[monomer]]ic GFP, which is often achieved by the dimer interface breaking A206K mutation. Wild-type GFP has a weak [[dimer]]ization tendency at concentrations above 5&nbsp;mg/mL. mGFP also stands for "modified GFP" which has been optimized through amino acid exchange for stable expression in plant cells.
 
==Structure==
GFP has a typical [[beta barrel]] structure, consisting of one β-sheet with alpha helix(s) containing the [[chromophore]] running through the center.<ref name=Ormo_1996>{{cite journal |author=Ormö M, Cubitt A, Kallio K, Gross L, Tsien R, Remington S |title=Crystal structure of the Aequorea victoria green fluorescent protein |journal=Science |volume=273 |issue=5280 |pages=1392–5 |year=1996 |pmid=8703075 |doi=10.1126/science.273.5280.1392}}</ref><ref name=Yang_1996>{{cite journal |author=Yang F, Moss L, Phillips G |title=The molecular structure of green fluorescent protein |journal=Nat Biotechnol |volume=14 |issue=10 |pages=1246–51 |year=1996 |pmid=9631087 |doi=10.1038/nbt1096-1246}}</ref> Inward facing sidechains of the barrel induce specific cyclization reactions in the tripeptide Ser65–Tyr66–Gly67 that lead to [[chromophore]] formation. This process of post-translational modification is referred to as ''maturation''. The hydrogen bonding network and electron stacking interactions with these sidechains influence the color of wtGFP and its numerous derivatives. The tightly packed nature of the barrel excludes solvent molecules, protecting the [[chromophore]] fluorescence from quenching by water.
 
==Use==
 
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.<ref name=Yutse_2005>{{cite journal |author=Yuste R |title=Fluorescence microscopy today |journal=Nat Methods |volume=2 |issue=12 |pages=902–4 |year=2005 |pmid=16299474 |doi=10.1038/nmeth1205-902}}</ref> While most small fluorescent molecules such as [[Fluorescein|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. For example, GFP had been widely used in labelling the [[Spermatozoon|spermatozoa]] of various organisms for identification purposes as in ''[[Drosophila melanogaster]]'', where expression of GFP can be used as a marker for a particular characteristic. GFP can also be expressed in different structures enabling morphological distinction. In such cases, the gene for the production of GFP is spliced into the genome of the organism in the region of the DNA which codes for the target proteins, and which is controlled by the same [[regulatory sequence]]; that is the gene's regulatory sequence now controls the production of GFP, in addition to the tagged protein(s). In cells where the gene is expressed, and the tagged proteins are produced, GFP is produced at the same time. Thus, only those cells in which the tagged gene is expressed, or the target proteins are produced, will fluoresce when observed under fluorescence microscopy. 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.
 
The [[Vertico SMI]] microscope using the SPDM Phymod technology uses the so-called "reversible photobleaching" effect of fluorescent dyes like GFP and its derivatives to localize them as single molecules in an optical resolution of 10&nbsp;nm. This can also be performed as a co-localization of two GFP derivatives (2CLM).<ref name="pmid19548231">{{cite journal | author = Gunkel M, Erdel F, Rippe K, Lemmer P, Kaufmann R, Hörmann C, Amberger R, Cremer C | title = Dual color localization microscopy of cellular nanostructures | journal = Biotechnol J | volume = 4 | issue = 6 | pages = 927–38 | year = 2009 | month = June | pmid = 19548231 | doi = 10.1002/biot.200900005 | url = | issn = }}</ref>
 
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).<ref name=Chudakov_2005>{{cite journal |author=Chudakov D, Lukyanov S, Lukyanov K |title=Fluorescent proteins as a toolkit for in vivo imaging |journal=Trends Biotechnol |volume=23 |issue=12 |pages=605–13 |year=2005 |pmid=16269193 |doi=10.1016/j.tibtech.2005.10.005}}</ref> Genetically combining several spectral variants of GFP is a useful trick for the analysis of brain circuitry ([[Brainbow]]).<ref name="pmid17972876">{{cite journal | author = Livet J, Weissman TA, Kang H, Draft RW, Lu J, Bennis RA, Sanes JR, Lichtman JW | title = Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system | journal = Nature | volume = 450 | issue = 7166 | pages = 56–62 | year = 2007 | month = November | pmid = 17972876 | doi = 10.1038/nature06293 | url = | issn = }}</ref> Other interesting uses of fluorescent proteins in the literature include using FPs as sensors of [[neuron]] [[membrane potential]],<ref name="pmid18679801">{{cite journal | author = Baker BJ, Mutoh H, Dimitrov D, Akemann W, Perron A, Iwamoto Y, Jin L, Cohen LB, Isacoff EY, Pieribone VA, Hughes T, Knöpfel T | title = Genetically encoded fluorescent sensors of membrane potential | journal = Brain Cell Biol | volume = 36 | issue = 1-4 | pages = 53–67 | year = 2008 | month = August | pmid = 18679801 | pmc = 2775812 | doi = 10.1007/s11068-008-9026-7 | url = | issn = }}</ref> tracking of [[AMPA]] receptors on cell membranes,<ref name="pmid16364901">{{cite journal | author = Adesnik H, Nicoll RA, England PM | title = Photoinactivation of native AMPA receptors reveals their real-time trafficking | journal = Neuron | volume = 48 | issue = 6 | pages = 977–85 | year = 2005 | month = December | pmid = 16364901 | doi = 10.1016/j.neuron.2005.11.030 | url = | issn = }}</ref> [[viral entry]] and the infection of individual [[influenza]] viruses and lentiviral viruses,<ref name="pmid12883000">{{cite journal | author = Lakadamyali M, Rust MJ, Babcock HP, Zhuang X | title = Visualizing infection of individual influenza viruses | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 100 | issue = 16 | pages = 9280–5 | year = 2003 | month = August | pmid = 12883000 | pmc = 170909 | doi = 10.1073/pnas.0832269100 | url = | issn = }}</ref><ref name="pmid18480844">{{cite journal | author = Joo KI, Wang P | title = Visualization of targeted transduction by engineered lentiviral vectors | journal = Gene Ther. | volume = 15 | issue = 20 | pages = 1384–96 | year = 2008 | month = October | pmid = 18480844 | pmc = 2575058 | doi = 10.1038/gt.2008.87 | url = | issn = }}</ref> etc.
 
It has also been found that new lines of transgenic GFP rats can be relevant for gene therapy as well as regenerative medicine.<ref name="pmid20094912">{{cite journal | author = Remy S, Tesson L, Usal C, Menoret S, Bonnamain V, Nerriere-Daguin V, Rossignol J, Boyer C, Nguyen TH, Naveilhan P, Lescaudron L, Anegon I | title = New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy | journal = Transgenic Res | volume = | issue = | pages = | year = 2010 | month = January | pmid = 20094912 | doi = 10.1007/s11248-009-9352-2 | url = | issn = }}</ref> By using "high-expresser" GFP transgenic rats display high expression in most tissues,and many cells that have not or only have been poorly characterized in previous GFP-transgenic rats. Through its ability to form internal chromophore without requiring accessory cofactors, enzymes or substrates other than molecular oxygen, GFP makes for an excellent tool in all forms of biology.<ref name="pmid18691124">{{cite journal | author = Stepanenko OV, Verkhusha VV, Kuznetsova IM, Uversky VN, Turoverov KK | title = Fluorescent proteins as biomarkers and biosensors: throwing color lights on molecular and cellular processes | journal = Curr. Protein Pept. Sci. | volume = 9 | issue = 4 | pages = 338–69 | year = 2008 | month = August | pmid = 18691124 | doi = | url = | issn = }}</ref>
 
==GFP in nature==
The purpose of both [[bioluminescence]] and GFP [[fluorescence]] in jellyfish is unknown. GFP is co-expressed with [[aequorin]] in small granules around the rim of the jellyfish bell. The secondary excitation peak (480&nbsp;nm) of GFP does absorb some of the blue emission of [[aequorin]], giving the [[bioluminescence]] a more green hue. The serine 65 residue of the GFP [[chromophore]] is responsible for the dual peaked excitation spectra of wild type GFP. It is conserved in all three GFP isoforms originally cloned by Prasher. Nearly all mutations of this residue consolidate the excitation spectra to a single peak at either 395&nbsp;nm or 480&nbsp;nm. The precise mechanism of this sensitivity is complex, but probably involves donation of a hydrogen from serine 65 to glutamate 222, which influences chromophore ionization.<ref name="Tsien_1998"/> Since a single mutation can dramatically enhance the 480&nbsp;nm excitation peak, making GFP a much more efficient partner of aequorin, ''A. victoria'' appears to evolutionarily prefer the less-efficient, dual peaked excitation spectrum. Roger Tsien has speculated that varying hydrostatic pressure with depth may effect serine 65's ability to donate a hydrogen to the chromophore and shift the ratio of the two excitation peaks. Thus the jellyfish may change the color of its bioluminescence with depth. Unfortunately, a collapse in the population of jellyfish in [[Friday Harbor, Washington|Friday Harbor]], where GFP was originally discovered, has hampered further study of the role of GFP in the jellyfish's natural environment.
 
==GFP in fine art==
[[Image:Steel Jellyfish (GFP).jpg|thumb|right|[[Julian Voss-Andreae|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.]]
 
[[Julian Voss-Andreae]], a German-born artist specializing in "protein sculptures,"<ref>{{cite journal | last = Voss-Andreae| first = J | year = 2005 | title = Protein Sculptures: Life's Building Blocks Inspire Art | journal = Leonardo | volume = 38 | pages = 41&ndash;45 | doi = 10.1162/leon.2005.38.1.41}}</ref> created sculptures based on the structure of GFP, including the 5'6" (1.70 m) tall "Green Fluorescent Protein" (2004)<ref>{{cite journal | last = Pawlak | first = Alexander | year = 2005 | title = Inspirierende Proteine | journal = Physik Journal | volume = 4 | pages = 12}}</ref> and the 4'7" (1.40 m) 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]].<ref>{{cite web | title = Julian Voss-Andreae Sculpture | url = http://www.julianvossandreae.com/ | accessdate = 2007-06-14}}</ref>
 
[[Eduardo Kac]] has also done some work with GFP, most notably "[[GFP Bunny]].<ref>{{cite web | title = GFP Bunny | url = http://www.ekac.org/gfpbunny.html#gfpbunnyanchor }}</ref>" Kac commissioned a French laboratory to create a green-fluorescent rabbit which is the subject of a series of his art pieces.
 
==Transgenic pets==
 
[[Alba (rabbit)|Alba]], a fluorescent rabbit, was commissioned by [[Eduardo Kac]] using GFP for purposes of art and social commentary.<ref>{{cite web|url=http://www.ekac.org/gfpbunny.html#gfpbunnyanchor|author=Eduardo Kac|title=GFP Bunny}}</ref> The US company Yorktown Technologies markets to aquarium shops green fluorescent [[zebrafish]] ([[GloFish]]) that were initially developed to detect pollution in waterways. NeonPets, a US based company markets green fluorescent mice to the pet industry as NeonMice.<ref>[http://www.neonmice.com] Glow-In-The Dark NeonMice</ref> Green fluorescent pigs, known as Noels were bred by a group of researchers led by Wu Shinn-Chih at the Department of Animal Science and Technology at [[National Taiwan University]].<ref>[http://news.bbc.co.uk/1/hi/world/asia-pacific/4605202.stm Scientists in Taiwan breed fluorescent green pigs]</ref>
 
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