:Green fluorescent protein

Image:Aequorea victoria.jpg

File:Neural progenitors in the olfactory bulb.tif

In the 1960s and 1970s, GFP, along with the separate luminescent protein aequorin (an enzyme that catalyzes the breakdown of luciferin, releasing light), was first purified from the jellyfish Aequorea victoria and its properties studied by Osamu Shimomura.{{cite journal | vauthors = Shimomura O, Johnson FH, Saiga Y | title = Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea | journal = Journal of Cellular and Comparative Physiology | volume = 59 | issue = 3 | pages = 223–39 | date = Jun 1962 | pmid = 13911999 | doi = 10.1002/jcp.1030590302 }} 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.{{cite journal | vauthors = Morise H, Shimomura O, Johnson FH, Winant J | title = Intermolecular energy transfer in the bioluminescent system of Aequorea | journal = Biochemistry | volume = 13 | issue = 12 | pages = 2656–62 | date = Jun 1974 | pmid = 4151620 | doi = 10.1021/bi00709a028 }} However, its utility as a tool for molecular biologists did not begin to be realized until 1992 when Douglas Prasher reported the cloning and nucleotide sequence of wtGFP in Gene.{{cite journal | vauthors = Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ | title = Primary structure of the Aequorea victoria green-fluorescent protein | journal = Gene | volume = 111 | issue = 2 | pages = 229–33 | date = Feb 1992 | pmid = 1347277 | doi = 10.1016/0378-1119(92)90691-H }} 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 wtGFP, with the first few amino acids deleted, in heterologous cells of E. coli and C. elegans, publishing the results in Science in 1994.{{cite journal | vauthors = Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC | s2cid = 9043327 | title = Green fluorescent protein as a marker for gene expression | journal = Science | volume = 263 | issue = 5148 | pages = 802–5 | date = Feb 1994 | pmid = 8303295 | doi = 10.1126/science.8303295 | bibcode = 1994Sci...263..802C }} Frederick Tsuji's lab independently reported the expression of the recombinant protein one month later.{{cite journal | vauthors = Inouye S, Tsuji FI | title = Aequorea green fluorescent protein. Expression of the gene and fluorescence characteristics of the recombinant protein | journal = FEBS Letters | volume = 341 | issue = 2–3 | pages = 277–80 | date = Mar 1994 | pmid = 8137953 | doi = 10.1016/0014-5793(94)80472-9 | doi-access = free | bibcode = 1994FEBSL.341..277I }} 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 {{convert|37|C}}.

The first reported crystal structure of a GFP was that of the S65T mutant by the Remington group in Science in 1996. One month later, the Phillips group independently reported the wild-type GFP structure in Nature Biotechnology. 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. Further research into GFP has shown that it is resistant to detergents, proteases, guanidinium chloride (GdmCl) treatments, and drastic temperature changes.{{cite journal | vauthors = Brejc K, Sixma TK, Kitts PA, Kain SR, Tsien RY, Ormö M, Remington SJ | title = Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 94 | issue = 6 | pages = 2306–2311 | date = March 1997 | pmid = 9122190 | pmc = 20083 | doi = 10.1073/pnas.94.6.2306 | bibcode = 1997PNAS...94.2306B | doi-access = free }}

Image:FPbeachTsien.jpg

Due to the potential for widespread usage and the evolving needs of researchers, many different mutants of GFP have been engineered.{{cite journal | vauthors = Shaner NC, Steinbach PA, Tsien RY | title = A guide to choosing fluorescent proteins | journal = Nature Methods | volume = 2 | issue = 12 | pages = 905–9 | date = Dec 2005 | pmid = 16299475 | doi = 10.1038/nmeth819 | s2cid = 10024284 | url = http://tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Methods%20-%20Choosing%20fluorescent%20proteins.pdf }}{{cite book | vauthors = Wilhelmsson M, Tor Y | title = Fluorescent Analogs of Biomolecular Building Blocks: Design and Applications | publisher = Wiley | location = New Jersey | year = 2016 | isbn = 978-1-118-17586-6 }} The first major improvement was a single point mutation (S65T) reported in 1995 in Nature by Roger Tsien.{{cite journal | vauthors = Heim R, Cubitt AB, Tsien RY | title = Improved green fluorescence | journal = Nature | volume = 373 | issue = 6516 | pages = 663–4 | date = Feb 1995 | pmid = 7854443 | doi = 10.1038/373663b0 | url = http://tsienlab.ucsd.edu/Publications/Heim%201995%20Nature%20-%20Improved%20GFP.PDF | bibcode = 1995Natur.373..663H | s2cid = 40179694 }} This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostability, and a shift of the major excitation peak to 488 nm, 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. A 37 °C folding efficiency (F64L) point mutant to this scaffold, yielding enhanced GFP (EGFP), was discovered in 1995 by the laboratories of Thastrup{{ cite patent | country = US | number = 6172188 | status = patent | title = Fluorescent Proteins | pubdate = 2001-01-09 | fdate = 1997-03-17 | pridate = 1995-09-22 | inventor = Thastrup O, Tullin S, Kongsbak Poulsen L, Bjørn S }} and Falkow.{{cite journal | vauthors = Cormack BP, Valdivia RH, Falkow S | title = FACS-optimized mutants of the green fluorescent protein (GFP) | journal = Gene | volume = 173 | issue = 1 Spec No | pages = 33–38 | date = 1996 | pmid = 8707053 | doi = 10.1016/0378-1119(95)00685-0 | doi-access = free }} EGFP allowed the practical use of GFPs in mammalian cells. EGFP has an extinction coefficient (denoted ε) of 55,000 M⁻¹cm⁻¹.{{cite journal | vauthors = McRae SR, Brown CL, Bushell GR | title = Rapid purification of EGFP, EYFP, and ECFP with high yield and purity | journal = Protein Expression and Purification | volume = 41 | issue = 1 | pages = 121–127 | date = May 2005 | pmid = 15802229 | doi = 10.1016/j.pep.2004.12.030 }} The fluorescence quantum yield (QY) of EGFP is 0.60. The relative brightness, expressed as ε•QY, is 33,000 M⁻¹cm⁻¹.

Superfolder GFP (sfGFP), a series of mutations that allow GFP to rapidly fold and mature even when fused to poorly folding peptides, was reported in 2006.{{cite journal | vauthors = Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS | title = Engineering and characterization of a superfolder green fluorescent protein | journal = Nature Biotechnology | volume = 24 | issue = 1 | pages = 79–88 | date = Jan 2006 | pmid = 16369541 | doi = 10.1038/nbt1172 | s2cid = 2966399 }}

Many other mutations have been made, including color mutants; in particular, blue fluorescent protein (EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein (ECFP, Cerulean, CyPet, mTurquoise2), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet). BFP derivatives (except mKalama1) contain the Y66H substitution. They exhibit a broad absorption band in the ultraviolet centered close to 380 nanometers and an emission maximum at 448 nanometers. A green fluorescent protein mutant (BFPms1) that preferentially binds Zn(II) and Cu(II) has been developed. BFPms1 have several important mutations including and the BFP chromophore (Y66H),Y145F for higher quantum yield, H148G for creating a hole into the beta-barrel and several other mutations that increase solubility. Zn(II) binding increases fluorescence intensity, while Cu(II) binding quenches fluorescence and shifts the absorbance maximum from 379 to 444 nm. Therefore, they can be used as a Zn biosensor.{{cite journal|author-link4=Elizabeth D. Getzoff | vauthors = Barondeau DP, Kassmann CJ, Tainer JA, Getzoff ED | title = Structural chemistry of a green fluorescent protein Zn biosensor | journal = Journal of the American Chemical Society | volume = 124 | issue = 14 | pages = 3522–3524 | date = Apr 2002 | pmid = 11929238 | doi = 10.1021/ja0176954 | bibcode = 2002JAChS.124.3522B }}

Image:174-GFPLikeProteins GFP-like Proteins.tif

More color variants are possible via chromophore binding. 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. In ECFP and Cerulean, the N-terminal half of the seventh strand exhibits two conformations. These conformations both have a complex set of van der Waals interactions with the chromophore. The Y145A and H148D mutations in Cerulean stabilize these interactions and allow the chromophore to be more planar, better packed, and less prone to collisional quenching.{{cite journal | vauthors = Lelimousin M, Noirclerc-Savoye M, Lazareno-Saez C, Paetzold B, Le Vot S, Chazal R, Macheboeuf P, Field MJ, Bourgeois D, Royant A | title = Intrinsic dynamics in ECFP and Cerulean control fluorescence quantum yield | journal = Biochemistry | volume = 48 | issue = 42 | pages = 10038–10046 | date = Oct 2009 | pmid = 19754158 | doi = 10.1021/bi901093w }}

Additional site-directed random mutagenesis in combination with fluorescence lifetime based screening has further stabilized the seventh β-strand resulting in a bright variant, mTurquoise2, with a quantum yield (QY) of 0.93.{{cite journal | vauthors = Goedhart J, von Stetten D, Noirclerc-Savoye M, Lelimousin M, Joosen L, Hink MA, van Weeren L, Gadella TW, Royant A | title = Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93% | journal = Nature Communications | volume = 3 | pages = 751 | date = 2012 | pmid = 22434194 | pmc = 3316892 | doi = 10.1038/ncomms1738 | bibcode = 2012NatCo...3..751G }} 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. These two classes of spectral variants are often employed for Förster 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-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.{{cite journal | vauthors = Miesenböck G, De Angelis DA, Rothman JE | title = Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins | journal = Nature | volume = 394 | issue = 6689 | pages = 192–5 | date = Jul 1998 | pmid = 9671304 | doi = 10.1038/28190 | bibcode = 1998Natur.394..192M | s2cid = 4320849 }}

Redox sensitive GFP (roGFP) was engineered by introduction of cysteines into the beta barrel structure. The redox state of the cysteines determines the fluorescent properties of roGFP.{{cite journal | vauthors = 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 = The Journal of Biological Chemistry | volume = 279 | issue = 13 | pages = 13044–53 | date = Mar 2004 | pmid = 14722062 | doi = 10.1074/jbc.M312846200 | doi-access = free }}

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.{{cite journal | vauthors = Zacharias DA, Violin JD, Newton AC, Tsien RY | s2cid = 14957077 | title = Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells | journal = Science | volume = 296 | issue = 5569 | pages = 913–16 | date = May 2002 | pmid = 11988576 | doi = 10.1126/science.1068539 | bibcode = 2002Sci...296..913Z }} 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.

File:Lancelet GFP GIF.gif

File:Pone.0011517.g001.png mimocerami]]

The purpose of both the (primary) bioluminescence (from aequorin's action on luciferin) and the (secondary) fluorescence of GFP 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 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 nm or 480 nm. The precise mechanism of this sensitivity is complex, but, it seems, involves donation of a hydrogen from serine 65 to glutamate 222, which influences chromophore ionization. Since a single mutation can dramatically enhance the 480 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 affect 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. However, a collapse in the population of jellyfish in Friday Harbor, where GFP was originally discovered, has hampered further study of the role of GFP in the jellyfish's natural environment.

Most species of lancelet are known to produce GFP in various regions of their body.{{cite journal | vauthors = Yue JX, Holland ND, Holland LZ, Deheyn DD | title = The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus) | journal = Scientific Reports | volume = 6 | issue = 1 | pages = 28350 | date = June 2016 | pmid = 27311567 | doi = 10.1038/srep28350 | pmc = 4911609 | bibcode = 2016NatSR...628350Y | doi-access = free }} Unlike A. victoria, lancelets do not produce their own blue light, and the origin of their endogenous GFP is still unknown. Some speculate that it attracts plankton towards the mouth of the lancelet, serving as a passive hunting mechanism. It may also serve as a photoprotective agent in the larvae, preventing damage caused by high-intensity blue light by converting it into lower-intensity green light. However, these theories have not been tested.

GFP-like proteins have been found in multiple species of marine copepods, particularly from the Pontellidae and Aetideidae families.{{cite journal | vauthors = Hunt ME, Scherrer MP, Ferrari FD, Matz MV | title = Very bright green fluorescent proteins from the Pontellid copepod Pontella mimocerami | journal = PLOS ONE | volume = 5 | issue = 7 | pages = e11517 | date = July 2010 | pmid = 20644720 | pmc = 2904364 | doi = 10.1371/journal.pone.0011517 | bibcode = 2010PLoSO...511517H | doi-access = free }} GFP isolated from Pontella mimocerami has shown high levels of brightness with a quantum yield of 0.92, making them nearly two-fold brighter than the commonly used EGFP isolated from A. victoria.{{cite web | title = eGFP | url = https://www.fpbase.org/protein/egfp/ | work = FPbase }}

File:Fluorescence from Fluorescent Proteins.jpg

There are many GFP-like proteins that, despite being in the same protein family as GFP, are not directly derived from Aequorea victoria. These include dsRed, eqFP611, Dronpa, TagRFPs, KFP, EosFP/IrisFP, Dendra, and so on. Having been developed from proteins in different organisms, these proteins can sometimes display unanticipated approaches to chromophore formation. Some of these, such as KFP, are developed from naturally non- or weakly-fluorescent proteins to be greatly improved upon by mutagenesis.{{cite journal | vauthors = Chudakov DM, Belousov VV, Zaraisky AG, Novoselov VV, Staroverov DB, Zorov DB, Lukyanov S, Lukyanov KA | title = Kindling fluorescent proteins for precise in vivo photolabeling | journal = Nature Biotechnology | volume = 21 | issue = 2 | pages = 191–4 | date = February 2003 | pmid = 12524551 | doi = 10.1038/nbt778 | s2cid = 52887792 }} When GFP-like barrels of different spectral characteristics are used, the excitation spectrum of one chromophore can be used to power another chromophore (FRET), allowing for conversion between wavelengths of light.{{cite journal | vauthors = Wiens MD, Shen Y, Li X, Salem MA, Smisdom N, Zhang W, Brown A, Campbell RE | title = A Tandem Green-Red Heterodimeric Fluorescent Protein with High FRET Efficiency | journal = ChemBioChem | volume = 17 | issue = 24 | pages = 2361–2367 | date = December 2016 | pmid = 27781394 | doi = 10.1002/cbic.201600492 | s2cid = 4301322 }}

FMN-binding fluorescent proteins (FbFPs) were developed in 2007 and are a class of small (11–16 kDa), oxygen-independent fluorescent proteins that are derived from blue-light receptors. They are intended especially for the use under anaerobic or hypoxic conditions, since the formation and binding of the flavin chromophore does not require molecular oxygen, as it is the case with the synthesis of the GFP chromophore.{{cite journal | vauthors = Drepper T, Eggert T, Circolone F, Heck A, Krauss U, Guterl JK, Wendorff M, Losi A, Gärtner W, Jaeger KE | title = Reporter proteins for in vivo fluorescence without oxygen | journal = Nature Biotechnology | volume = 25 | issue = 4 | pages = 443–445 | date = April 2007 | pmid = 17351616 | doi = 10.1038/nbt1293 | s2cid = 7335755 }}

File:White Light Image of Fluorescent Proteins.jpg

Fluorescent proteins with other chromophores, such as UnaG with bilirubin, can display unique properties like red-shifted emission above 600 nm or photoconversion from a green-emitting state to a red-emitting state. They can have excitation and emission wavelengths far enough apart to achieve conversion between red and green light.

A new class of fluorescent protein was engineered from α-allophycocyanin, a phycobiliprotein found in the cyanobacterium Trichodesmium erythraeum, and was named small ultra red fluorescent protein (smURFP) in 2016. smURFP autocatalytically incorporates the chromophore biliverdin without the need for an external protein known as a lyase.{{cite journal | vauthors = Rodriguez EA, Tran GN, Gross LA, Crisp JL, Shu X, Lin JY, Tsien RY | title = A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein | journal = Nature Methods | volume = 13 | issue = 9 | pages = 763–9 | date = September 2016 | pmid = 27479328 | pmc = 5007177 | doi = 10.1038/nmeth.3935 }}{{cite book | vauthors = Mattson S, Tran GN, Rodriguez EA |chapter=Directed Evolution of Fluorescent Proteins in Bacteria |date=2023 |title=Fluorescent Proteins |series=Methods in Molecular Biology |volume=2564 |pages=75–97 | veditors = Sharma M |place=New York, NY |publisher=Springer US |language=en |doi=10.1007/978-1-0716-2667-2_4 |isbn=978-1-0716-2666-5 |pmid=36107338 }} Jellyfish- and coral-derived GFP-like proteins require oxygen and produce a stoichiometric amount of hydrogen peroxide upon chromophore formation.{{cite journal | vauthors = Tsien RY | s2cid = 8138960 | title = The green fluorescent protein | journal = Annual Review of Biochemistry | volume = 67 | issue = 1 | pages = 509–44 | date = 1998-01-01 | pmid = 9759496 | doi = 10.1146/annurev.biochem.67.1.509 }} smURFP does not require oxygen or produce hydrogen peroxide. smURFP has a large extinction coefficient (180,000 M⁻¹ cm⁻¹) and has a modest quantum yield (0.20), which makes it comparable biophysical brightness to eGFP and ~2-fold brighter than most red or far-red fluorescent proteins derived from coral. smURFP spectral properties are similar to the organic dye Cy5.{{cite journal | vauthors = Maiti A, Buffalo CZ, Saurabh S, Montecinos-Franjola F, Hachey JS, Conlon WJ, Tran GN, Hassan B, Walters KJ, Drobizhev M, Moerner WE, Ghosh P, Matsuo H, Tsien RY, Lin JY, Rodriguez EA | title = Structural and photophysical characterization of the small ultra-red fluorescent protein | journal = Nature Communications | volume = 14 | issue = 1 | pages = 4155 | date = July 2023 | pmid = 37438348 | pmc = 10338489 | doi = 10.1038/s41467-023-39776-9 | bibcode = 2023NatCo..14.4155M }}

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Reviews on new classes of fluorescent proteins and applications can be found in the cited reviews.{{cite journal | vauthors = Rodriguez EA, Campbell RE, Lin JY, Lin MZ, Miyawaki A, Palmer AE, Shu X, Zhang J, Tsien RY | title = The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins | journal = Trends in Biochemical Sciences | volume = 42 | issue = 2 | pages = 111–129 | date = February 2017 | pmid = 27814948 | pmc = 5272834 | doi = 10.1016/j.tibs.2016.09.010 }}{{cite journal| vauthors = Montecinos-Franjola F, Lin JY, Rodriguez EA |date=2020-11-16|title=Fluorescent proteins for in vivo imaging, where's the biliverdin? |journal=Biochemical Society Transactions |volume=48|issue=6|pages=2657–2667 |doi=10.1042/BST20200444 |pmid=33196077|s2cid=226971864 }}

GFP has a beta barrel structure consisting of eleven β-strands with a pleated sheet arrangement, with an alpha helix containing the covalently bonded chromophore 4-(p-hydroxybenzylidene)imidazolidin-5-one (HBI) running through the center.{{cite journal | vauthors = Ormö M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ | s2cid = 43030290 | title = Crystal structure of the Aequorea victoria green fluorescent protein | journal = Science | volume = 273 | issue = 5280 | pages = 1392–5 | date = Sep 1996 | pmid = 8703075 | doi = 10.1126/science.273.5280.1392 | bibcode = 1996Sci...273.1392O }}{{cite journal | vauthors = Yang F, Moss LG, Phillips GN | title = The molecular structure of green fluorescent protein | journal = Nature Biotechnology | volume = 14 | issue = 10 | pages = 1246–51 | date = Oct 1996 | pmid = 9631087 | doi = 10.1038/nbt1096-1246 | url = https://scholarship.rice.edu/bitstream/1911/19233/1/9727628.PDF | hdl = 1911/19233 | s2cid = 34713931 | hdl-access = free }} Five shorter alpha helices form caps on the ends of the structure. The beta barrel structure is a nearly perfect cylinder, 42Å long and 24Å in diameter (some studies have reported a diameter of 30Å), creating what is referred to as a "β-can" formation, which is unique to the GFP-like family. HBI, the spontaneously modified form of the tripeptide Ser65–Tyr66–Gly67, is nonfluorescent in the absence of the properly folded GFP scaffold and exists mainly in the un-ionized phenol form in wtGFP.{{cite journal | vauthors = Bokman SH, Ward WW | title = Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein | journal = Biochemistry | volume = 21 | issue = 19 | pages = 4535–4540 | date = 1982 | doi = 10.1021/bi00262a003| pmid = 6128025 }} Inward-facing sidechains of the barrel induce specific cyclization reactions in Ser65–Tyr66–Gly67 that induce ionization of HBI to the phenolate form and chromophore formation. This process of post-translational modification is referred to as maturation.{{cite journal | vauthors = Pouwels LJ, Zhang L, Chan NH, Dorrestein PC, Wachter RM | title = Kinetic isotope effect studies on the de novo rate of chromophore formation in fast- and slow-maturing GFP variants | journal = Biochemistry | volume = 47 | issue = 38 | pages = 10111–22 | date = Sep 2008 | pmid = 18759496 | pmc = 2643082 | doi = 10.1021/bi8007164 }} The hydrogen-bonding network and electron-stacking interactions with these sidechains influence the color, intensity and photostability of GFP and its numerous derivatives.{{cite journal | vauthors = Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA | s2cid = 10767597 | title = Fluorescent proteins and their applications in imaging living cells and tissues | journal = Physiological Reviews | volume = 90 | issue = 3 | pages = 1103–63 | date = Jul 2010 | pmid = 20664080 | doi = 10.1152/physrev.00038.2009 }} The tightly packed nature of the barrel excludes solvent molecules, protecting the chromophore fluorescence from quenching by water. In addition to the auto-cyclization of the Ser65-Tyr66-Gly67, a 1,2-dehydrogenation reaction occurs at the Tyr66 residue. Besides the three residues that form the chromophore, residues such as Gln94, Arg96, His148, Thr203, and Glu222 all act as stabilizers. The residues of Gln94, Arg96, and His148 are able to stabilize by delocalizing the chromophore charge. Arg96 is the most important stabilizing residue due to the fact that it prompts the necessary structural realignments that are necessary from the HBI ring to occur. Any mutation to the Arg96 residue would result in a decrease in the development rate of the chromophore because proper electrostatic and steric interactions would be lost. Tyr66 is the recipient of hydrogen bonds and does not ionize in order to produce favorable electrostatics.{{cite journal | vauthors = Stepanenko OV, Verkhusha VV, Shavlovsky MM, Kuznetsova IM, Uversky VN, Turoverov KK | title = Understanding the role of Arg96 in structure and stability of green fluorescent protein | journal = Proteins | volume = 73 | issue = 3 | pages = 539–551 | date = November 2008 | pmid = 18470931 | pmc = 2908022 | doi = 10.1002/prot.22089 }}

File:GFP Fluorescent Protein Movie.gif

File:Final Y66 and Y145 1EMA structure of GFP.png

Blue fluorescent protein (BFP) is the blue variant of green fluorescent protein (GFP). BFP has a very similar structure to GFP. In the BFP structure, two substitution mutations in the amino acid sequence change its fluorescence from green to blue. The first mutation occurs inside the chromophore of GFP at position 66 which changes a tyrosine to a histidine. The other mutation in BFP is on the tyrosine at position 145 which mutates to phenylalanine. The autocatalytic cyclization and oxidation of the serine, tyrosine, and glycine form the GFP chromophore. These three residues at positions 65-67 make up the green fluorescent chromophore. When the tyrosine in the chromophore is substituted by a histidine, it changes the folding structure of the protein and emission spectra. The T145F mutation is also added to increase the stability of the protein and well as intensify the fluorescence. These mutations are what change GFP to BFP.

{{hatnote|For a synthetic analogue see also 3,5-Difluoro-4-hydroxybenzylidene imidazolinone.}}

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Mechanistically, the process involves base-mediated cyclization followed by dehydration and oxidation. In the reaction of 7a to 8 involves the formation of an enamine from the imine, while in the reaction of 7b to 9 a proton is abstracted.{{cite journal | vauthors = Rosenow MA, Huffman HA, Phail ME, Wachter RM | title = The crystal structure of the Y66L variant of green fluorescent protein supports a cyclization-oxidation-dehydration mechanism for chromophore maturation | journal = Biochemistry | volume = 43 | issue = 15 | pages = 4464–4472 | date = April 2004 | pmid = 15078092 | doi = 10.1021/bi0361315 }} The formed HBI fluorophore is highlighted in green.

The reactions are catalyzed by residues Glu222 and Arg96.{{Cite journal| vauthors = Ma Y, Yu JG, Sun Q, Li Z, Smith SC |date=2015-07-01|title=The mechanism of dehydration in chromophore maturation of wild-type green fluorescent protein: A theoretical study |journal=Chemical Physics Letters|language=en|volume=631-632|pages=42–46|doi=10.1016/j.cplett.2015.04.061|bibcode=2015CPL...631...42M |issn=0009-2614}} An analogous mechanism is also possible with threonine in place of Ser65.

Green fluorescent protein may be used as a reporter gene.{{cite journal | vauthors = Jugder BE, Welch J, Braidy N, Marquis CP | title = Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein) | journal = PeerJ | volume = 4 | pages = e2269 | date = 2016-07-26 | pmid = 27547572 | pmc = 4974937 | doi = 10.7717/peerj.2269 | doi-access = free }}{{cite journal | vauthors = Arun KH, Kaul CL, Ramarao P | title = Green fluorescent proteins in receptor research: an emerging tool for drug discovery | journal = Journal of Pharmacological and Toxicological Methods | volume = 51 | issue = 1 | pages = 1–23 | year = 2005 | pmid = 15596111 | doi = 10.1016/j.vascn.2004.07.006 }}

For example, GFP can be used as a reporter for environmental toxicity levels. This protein has been shown to be an effective way to measure the toxicity levels of various chemicals including ethanol, p-formaldehyde, phenol, triclosan, and paraben. GFP is great as a reporter protein because it has no effect on the host when introduced to the host's cellular environment. Due to this ability, no external visualization stain, ATP, or cofactors are needed. With regards to pollutant levels, the fluorescence was measured in order to gauge the effect that the pollutants have on the host cell. The cellular density of the host cell was also measured. Results from the study conducted by Song, Kim, & Seo (2016) showed that there was a decrease in both fluorescence and cellular density as pollutant levels increased. This was indicative of the fact that cellular activity had decreased. More research into this specific application in order to determine the mechanism by which GFP acts as a pollutant marker.{{cite journal | vauthors = Song YH, Kim CS, Seo JH | title = Noninvasive monitoring of environmental toxicity through green fluorescent protein expressing Escherichia coli. | journal = Korean Journal of Chemical Engineering | date = April 2016 | volume = 33 | issue = 4 | pages = 1331–6 | doi = 10.1007/s11814-015-0253-1 | s2cid = 62828580 }} Similar results have been observed in zebrafish because zebrafish that were injected with GFP were approximately twenty times more susceptible to recognize cellular stresses than zebrafish that were not injected with GFP.{{cite journal | vauthors = Pan Y, Leifert A, Graf M, Schiefer F, Thoröe-Boveleth S, Broda J, Halloran MC, Hollert H, Laaf D, Simon U, Jahnen-Dechent W | title = High-sensitivity real-time analysis of nanoparticle toxicity in green fluorescent protein-expressing zebrafish | journal = Small | location = Weinheim an Der Bergstrasse, Germany | volume = 9 | issue = 6 | pages = 863–9 | date = March 2013 | pmid = 23143852 | doi = 10.1002/smll.201201173 }}

The biggest advantage of GFP is that it can be heritable, depending on how it was introduced, allowing for continued study of cells and tissues it is expressed in. Visualizing GFP is noninvasive, requiring only illumination with blue light. GFP alone does not interfere with biological processes, but when fused to proteins of interest, careful design of linkers is required to maintain the function of the protein of interest. Moreover, if used with a monomer it is able to diffuse readily throughout cells.{{cite journal | vauthors = Chalfie M | title = GFP: Lighting up life | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 106 | issue = 25 | pages = 10073–10080 | date = Jun 2009 | pmid = 19553219 | doi = 10.1073/pnas.0904061106 | pmc=2700921| bibcode = 2009PNAS..10610073C | doi-access = free }}

{{Main|Fluorescence microscope}}

Image:GFP Superresolution Christoph Cremer.JPGs (GFP-Snf2H and RFP-H2A), Co-localisation studies (2CLM) in the nucleus of a bone cancer cell. 120.000 localized molecules in a widefield area (470 μm²).]]

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.{{cite journal | vauthors = Yuste R | title = Fluorescence microscopy today | journal = Nature Methods | volume = 2 | issue = 12 | pages = 902–4 | date = Dec 2005 | pmid = 16299474 | doi = 10.1038/nmeth1205-902 | s2cid = 205418407 }} 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.

There are many techniques to utilize GFP in a live cell imaging experiment. The most direct way of utilizing GFP is to directly attach it to a protein of interest. For example, GFP can be included in a plasmid expressing other genes to indicate a successful transfection of a gene of interest. Another method is to use a GFP that contains a mutation where the fluorescence will change from green to yellow over time, which is referred to as a fluorescent timer. With the fluorescent timer, researchers can study the state of protein production such as recently activated, continuously activated, or recently deactivated based on the color reported by the fluorescent protein.{{cite journal | vauthors = Terskikh A, Fradkov A, Ermakova G, Zaraisky A, Tan P, Kajava AV, Zhao X, Lukyanov S, Matz M, Kim S, Weissman I, Siebert P | title = "Fluorescent timer": protein that changes color with time | journal = Science | volume = 290 | issue = 5496 | pages = 1585–8 | date = November 2000 | pmid = 11090358 | doi = 10.1126/science.290.5496.1585 | bibcode = 2000Sci...290.1585T }} In yet another example, scientists have modified GFP to become active only after exposure to irradiation giving researchers a tool to selectively activate certain portions of a cell and observe where proteins tagged with the GFP move from the starting location.{{cite journal | vauthors = Patterson GH, Lippincott-Schwartz J | title = A photoactivatable GFP for selective photolabeling of proteins and cells | journal = Science | volume = 297 | issue = 5588 | pages = 1873–7 | date = September 2002 | pmid = 12228718 | doi = 10.1126/science.1074952 | bibcode = 2002Sci...297.1873P | s2cid = 45058411 }} These are only two examples in a burgeoning field of fluorescent microcopy and a more complete review of biosensors utilizing GFP and other fluorescent proteins can be found here {{cite journal | vauthors = Lin W, Mehta S, Zhang J | title = Genetically encoded fluorescent biosensors illuminate kinase signaling in cancer | journal = The Journal of Biological Chemistry | volume = 294 | issue = 40 | pages = 14814–14822 | date = October 2019 | pmid = 31434714 | pmc = 6779441 | doi = 10.1074/jbc.REV119.006177 | doi-access = free }}

For example, GFP had been widely used in labelling the 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 incorporated into the genome of the organism in the region of the DNA that codes for the target proteins and that 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. Obtained data are also used to calibrate mathematical models of intracellular systems and to estimate rates of gene expression.{{cite journal | vauthors = Komorowski M, Finkenstädt B, Rand D | title = Using a single fluorescent reporter gene to infer half-life of extrinsic noise and other parameters of gene expression | journal = Biophysical Journal | volume = 98 | issue = 12 | pages = 2759–2769 | date = Jun 2010 | pmid = 20550887 | pmc = 2884236 | doi = 10.1016/j.bpj.2010.03.032 | bibcode = 2010BpJ....98.2759K }} Similarly, GFP can be used as an indicator of protein expression in heterologous systems. In this scenario, fusion proteins containing GFP are introduced indirectly, using RNA of the construct, or directly, with the tagged protein itself. This method is useful for studying structural and functional characteristics of the tagged protein on a macromolecular or single-molecule scale with fluorescence microscopy.

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 nm. This can also be performed as a co-localization of two GFP derivatives (2CLM).{{cite journal | vauthors = 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 = Biotechnology Journal | volume = 4 | issue = 6 | pages = 927–38 | date = Jun 2009 | pmid = 19548231 | doi = 10.1002/biot.200900005 | s2cid = 18162278 | url = https://hal.archives-ouvertes.fr/hal-00494027/document }}

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).{{cite journal | vauthors = Chudakov DM, Lukyanov S, Lukyanov KA | title = Fluorescent proteins as a toolkit for in vivo imaging | journal = Trends in Biotechnology | volume = 23 | issue = 12 | pages = 605–13 | date = Dec 2005 | pmid = 16269193 | doi = 10.1016/j.tibtech.2005.10.005 }} GFP is considered to be a reliable reporter of gene expression in eukaryotic cells when the fluorescence is measured by flow cytometry.{{cite journal | vauthors = Soboleski MR, Oaks J, Halford WP | title = Green fluorescent protein is a quantitative reporter of gene expression in individual eukaryotic cells | journal = FASEB Journal | volume = 19 | issue = 3 | pages = 440–442 | date = March 2005 | pmid = 15640280 | pmc = 1242169 | doi = 10.1096/fj.04-3180fje | doi-access = free }} Genetically combining several spectral variants of GFP is a useful trick for the analysis of brain circuitry (Brainbow).{{cite journal | vauthors = 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 | date = Nov 2007 | pmid = 17972876 | doi = 10.1038/nature06293 | bibcode = 2007Natur.450...56L | s2cid = 4402093 }} Other interesting uses of fluorescent proteins in the literature include using FPs as sensors of neuron membrane potential,{{cite journal | vauthors = 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 Biology | volume = 36 | issue = 1–4 | pages = 53–67 | date = Aug 2008 | pmid = 18679801 | pmc = 2775812 | doi = 10.1007/s11068-008-9026-7 }} tracking of AMPA receptors on cell membranes,{{cite journal | vauthors = 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 | date = Dec 2005 | pmid = 16364901 | doi = 10.1016/j.neuron.2005.11.030 | doi-access = free }} viral entry and the infection of individual influenza viruses and lentiviral viruses,{{cite journal | vauthors = Lakadamyali M, Rust MJ, Babcock HP, Zhuang X | title = Visualizing infection of individual influenza viruses | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 100 | issue = 16 | pages = 9280–5 | date = Aug 2003 | pmid = 12883000 | pmc = 170909 | doi = 10.1073/pnas.0832269100 | bibcode = 2003PNAS..100.9280L | doi-access = free }}{{cite journal | vauthors = Joo KI, Wang P | title = Visualization of targeted transduction by engineered lentiviral vectors | journal = Gene Therapy | volume = 15 | issue = 20 | pages = 1384–96 | date = Oct 2008 | pmid = 18480844 | pmc = 2575058 | doi = 10.1038/gt.2008.87 }} etc.

It has also been found that new lines of transgenic GFP rats can be relevant for gene therapy as well as regenerative medicine.{{cite journal | vauthors = 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 Research | volume = 19 | issue = 5 | pages = 745–63 | date = Oct 2010 | pmid = 20094912 | doi = 10.1007/s11248-009-9352-2 | s2cid = 42499768 }} By using "high-expresser" GFP, transgenic rats display high expression in most tissues, and many cells that have not been characterized or have been only poorly characterized in previous GFP-transgenic rats.

GFP has been shown to be useful in cryobiology as a viability assay. Correlation of viability as measured by trypan blue assays were 0.97.{{cite journal | vauthors = Elliott G, McGrath J, Crockett-Torabi E | title = Green fluorescent protein: A novel viability assay for cryobiological applications | journal = Cryobiology | volume = 40 | issue = 4 | pages = 360–369 | date = Jun 2000 | pmid = 10924267 | doi = 10.1006/cryo.2000.2258 }} Another application is the use of GFP co-transfection as internal control for transfection efficiency in mammalian cells.{{cite journal | vauthors = Fakhrudin N, Ladurner A, Atanasov AG, Heiss EH, Baumgartner L, Markt P, Schuster D, Ellmerer EP, Wolber G, Rollinger JM, Stuppner H, Dirsch VM | title = Computer-aided discovery, validation, and mechanistic characterization of novel neolignan activators of peroxisome proliferator-activated receptor gamma | journal = Molecular Pharmacology | volume = 77 | issue = 4 | pages = 559–66 | date = Apr 2010 | pmid = 20064974 | pmc = 3523390 | doi = 10.1124/mol.109.062141 }}

A novel possible use of GFP includes using it as a sensitive monitor of intracellular processes via an eGFP laser system made out of a human embryonic kidney cell line. The first engineered living laser is made by an eGFP expressing cell inside a reflective optical cavity and hitting it with pulses of blue light. At a certain pulse threshold, the eGFP's optical output becomes brighter and completely uniform in color of pure green with a wavelength of 516 nm. Before being emitted as laser light, the light bounces back and forth within the resonator cavity and passes the cell numerous times. By studying the changes in optical activity, researchers may better understand cellular processes.{{cite journal | vauthors = Gather MC, Yun SH | s2cid = 54971962 | title = Single-cell biological lasers | date = 2011 | journal = Nature Photonics | volume = 5 | issue = 7 | pages = 406–410 | doi = 10.1038/nphoton.2011.99 | bibcode = 2011NaPho...5..406G }}{{cite journal | vauthors = Matson J | date = 2011 | title = Green Fluorescent Protein Makes for Living Lasers | journal = Scientific American | url = http://www.scientificamerican.com/article.cfm?id=biological-laser-cell | access-date = 2011-06-13 }}

GFP is used widely in cancer research to label and track cancer cells. GFP-labelled cancer cells have been used to model metastasis, the process by which cancer cells spread to distant organs.{{cite journal | vauthors = Kouros-Mehr H, Bechis SK, Slorach EM, Littlepage LE, Egeblad M, Ewald AJ, Pai SY, Ho IC, Werb Z | title = GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model | journal = Cancer Cell | volume = 13 | issue = 2 | pages = 141–52 | date = Feb 2008 | pmid = 18242514 | pmc = 2262951 | doi = 10.1016/j.ccr.2008.01.011 }}

GFP can be used to analyse the colocalization of proteins. This is achieved by "splitting" the protein into two fragments which are able to self-assemble, and then fusing each of these to the two proteins of interest. Alone, these incomplete GFP fragments are unable to fluoresce. However, if the two proteins of interest colocalize, then the two GFP fragments assemble together to form a GFP-like structure which is able to fluoresce. Therefore, by measuring the level of fluorescence it is possible to determine whether the two proteins of interest colocalize.{{cite journal | vauthors = Cabantous S, Terwilliger TC, Waldo GS | title = Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein | journal = Nature Biotechnology | volume = 23 | issue = 1 | pages = 102–7 | date = January 2005 | pmid = 15580262 | doi = 10.1038/nbt1044 | s2cid = 25833063 }}

Macro-scale biological processes, such as the spread of virus infections, can be followed using GFP labeling.{{cite journal | vauthors = Rodman MK, Yadav NS, Artus NN | title = Progression of geminivirus-induced transgene silencing is associated with transgene methylation | journal = The New Phytologist | volume = 155 | issue = 3 | pages = 461–468 | date = September 2002 | pmid = 33873315 | doi = 10.1046/j.1469-8137.2002.00467.x | bibcode = 2002NewPh.155..461R }} In the past, mutagenic ultra violet light (UV) has been used to illuminate living organisms (e.g., see{{cite journal | vauthors = Zhu YJ, Agbayani R, Moore PH | title = Green fluorescent protein as a visual selection marker for papaya (Carica papaya L.) transformation | journal = Plant Cell Reports | volume = 22 | issue = 9 | pages = 660–7 | date = Apr 2004 | pmid = 14749892 | doi = 10.1007/s00299-004-0755-5 | s2cid = 23198182 }}) to detect and photograph the GFP expression. Recently, a technique using non-mutagenic LED lights{{cite journal | vauthors = Niwa Y, Hirano T, Yoshimoto K, Shimizu M, Kobayashi H | s2cid = 292648 | title = Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants | journal = The Plant Journal | volume = 18 | issue = 4 | pages = 455–63 | year = 1999 | pmid = 10406127 | doi = 10.1046/j.1365-313X.1999.00464.x | doi-access = free }} have been developed for macro-photography.{{cite journal | vauthors = Baker SS, Vidican CB, Cameron DS, Greib HG, Jarocki CC, Setaputri AW, Spicuzza CH, Burr AA, Waqas MA, Tolbert DA | title = An epifluorescent attachment improves whole-plant digital photography of Arabidopsis thaliana expressing red-shifted green fluorescent protein | journal = AoB Plants | volume = 2012 | pages = pls003 | date = 2012-01-01 | pmid = 22479674 | pmc = 3296078 | doi = 10.1093/aobpla/pls003 }} The technique uses an epifluorescence camera attachment{{cite web | url = http://planted.botany.org/index.php?P=FullRecord&ID=570 | title = PlantEdDL - Using SRL digital cameras in quantitative investigations of plants expressing green fluorescent protein (GFP) | website = planted.botany.org | access-date = 2016-03-23 }} based on the same principle used in the construction of epifluorescence microscopes.

Image:GFP Mice 01.jpg

Alba, a green-fluorescent rabbit, was created by a French laboratory commissioned by Eduardo Kac using GFP for purposes of art and social commentary.{{cite web|url= http://www.ekac.org/gfpbunny.html#gfpbunnyanchor | vauthors = Kac E |title=GFP Bunny}} 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 has marketed green fluorescent mice to the pet industry as NeonMice.{{cite web |url=http://neonmice.com/ |archive-url=https://web.archive.org/web/20090214004617/http://www.neonmice.com/ |archive-date=February 14, 2009 |title=Glow-In-The Dark NeonMice |url-status=dead |access-date=August 30, 2016}} 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.[http://news.bbc.co.uk/1/hi/world/asia-pacific/4605202.stm Scientists in Taiwan breed fluorescent green pigs] A Japanese-American Team created green-fluorescent cats as proof of concept to use them potentially as model organisms for diseases, particularly HIV.{{cite journal|author-link5=Eric Poeschla | vauthors = Wongsrikeao P, Saenz D, Rinkoski T, Otoi T, Poeschla E | title = Antiviral restriction factor transgenesis in the domestic cat | journal = Nature Methods | volume = 8 | issue = 10 | pages = 853–9 | date = 2011 | pmid = 21909101 | pmc = 4006694 | doi = 10.1038/nmeth.1703 }} In 2009 a South Korean team from Seoul National University bred the first transgenic beagles with fibroblast cells from sea anemones. The dogs give off a red fluorescent light, and they are meant to allow scientists to study the genes that cause human diseases like narcolepsy and blindness.{{Cite web | vauthors = Callaway E | date = 23 April 2009 | url=https://www.newscientist.com/article/dn17003-fluorescent-puppy-is-worlds-first-transgenic-dog.html#.U3O8tvldV8G | title=Fluorescent puppy is world's first transgenic dog | work = New Scientist }}

Julian Voss-Andreae, a German-born artist specializing in "protein sculptures,"{{cite journal | vauthors = Voss-Andreae J | date = 2005 | title = Protein Sculptures: Life's Building Blocks Inspire Art | journal = Leonardo | volume = 38 | pages = 41–45 | doi = 10.1162/leon.2005.38.1.41| s2cid = 57558522 }} created sculptures based on the structure of GFP, including the {{convert|1.70|m|ftin|abbr=off}} tall "Green Fluorescent Protein" (2004){{cite journal | vauthors = Pawlak A | date = 2005 | title = Inspirierende Proteine | journal = Physik Journal | volume = 4 | pages = 12 | url = https://pro-physik.de/zeitschriften/download/16101 }} and the {{convert|1.40|m|ftin|abbr=off}} tall "Steel Jellyfish" (2006). The latter sculpture is located at the place of GFP's discovery by Shimomura in 1962, the University of Washington's Friday Harbor Laboratories.{{cite web | title = Julian Voss-Andreae Sculpture | url = http://www.julianvossandreae.com/ | access-date = 2007-06-14}}

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

• Protein tag
• pGLO
• Yellow fluorescent protein
• Genetically encoded voltage indicator

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{{Reflist|30em}}

{{refbegin}}

• {{cite book | vauthors = Pieribone V, Gruber D | title = Aglow in the Dark: The Revolutionary Science of Biofluorescence | publisher = Belknap Press | location = Cambridge | date = 2006 | isbn = 978-0-674-01921-8 | oclc = 60321612 | url-access = registration | url = https://archive.org/details/aglowindarkrevol00pier }} Popular science book describing history and discovery of GFP
• {{cite book | vauthors = Zimmer M | title = Glowing Genes: A Revolution In Biotechnology| publisher = Prometheus Books | location = Buffalo, NY | date = 2005 | isbn = 978-1-59102-253-4 | oclc = 56614624 }}

{{refend}}

{{Library resources box

|onlinebooks=no

|by=no

|lcheading=Green fluorescent protein}}

{{Commons category|Green fluorescent proteins}}

• [http://www.scholarpedia.org/article/Fluorescent_proteins A comprehensive article on fluorescent proteins at Scholarpedia]
• [http://www.nature.com/nmeth/focus/fluorescence/classics/proteins.html Brief summary of landmark GFP papers]
• Interactive Java applet demonstrating the chemistry behind the [https://web.archive.org/web/20130516164120/http://www.olympusconfocal.com/java/fpfluorophores/gfpfluorophore/index.html formation of the GFP chromophore]
• [http://nobelprize.org/mediaplayer/index.php?id=1070 Video of 2008 Nobel Prize lecture of Roger Tsien on fluorescent proteins]
• [http://tsienlab.ucsd.edu/Documents/REF%20-%20Fluorophore%20Spectra.xls Excitation and emission spectra for various fluorescent proteins]
• [http://www.rsc.org/Publishing/Journals/CS/Article.asp?Type=Issue&JournalCode=CS&Issue=10&Volume=38&SubYear=2009 Green Fluorescent Protein Chem Soc Rev themed issue] dedicated to the 2008 Nobel Prize winners in Chemistry, Professors Osamu Shimomura, Martin Chalfie and Roger Y. Tsien
• [//www.rcsb.org/pdb/101/motm.do?momID=42 Molecule of the Month, June 2003]: an illustrated overview of GFP by David Goodsell.
• [//www.rcsb.org/pdb/101/motm.do?momID=174 Molecule of the Month, June 2014]: an illustrated overview of GFP-like variants by David Goodsell.
• [https://www.fpbase.org/protein/1XF1B/ Green Fluorescent Protein] on FPbase, a fluorescent protein database
• {{PDBe-KB2|P42212|Green fluorescent protein}}

{{Protein methods}}

{{DEFAULTSORT:Green Fluorescent Protein}}

Category:Protein methods

Category:Recombinant proteins

Category:Cell imaging

Category:Protein imaging

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