Summary: Green fluorescent protein
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Green fluorescent protein Edit Wikipedia article
|Green fluorescent protein|
Structure of the Aequorea victoria green fluorescent protein.
The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues (26.9 kDa) that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the jellyfish Aequorea victoria. The GFP from A. victoria has a major excitation peak at a wavelength of 395 nm and a minor one at 475 nm. Its emission peak is at 509 nm, which is in the lower green portion of the visible spectrum. The fluorescence quantum yield (QY) of GFP is 0.79. The GFP from the sea pansy (Renilla reniformis) has a single major excitation peak at 498 nm. GFP makes for an excellent tool in many forms of biology due to its ability to form internal chromophore without requiring any accessory cofactors, gene products, or enzymes / substrates other than molecular oxygen.
In cell and molecular biology, the GFP gene is frequently used as a reporter of expression. It has been used in modified forms to make biosensors, and many animals have been created that express GFP, which demonstrates a proof of concept that a gene can be expressed throughout a given organism, in selected organs, or in cells of interest. GFP can be introduced into animals or other species through transgenic techniques, and maintained in their genome and that of their offspring. To date, GFP has been expressed in many species, including bacteria, yeasts, fungi, fish and mammals, including in human cells. Scientists Roger Y. Tsien, Osamu Shimomura, and Martin Chalfie were awarded the 2008 Nobel Prize in Chemistry on 10 October 2008 for their discovery and development of the green fluorescent protein.
Wild-type GFP (wtGFP)
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 Aequorea victoria and its properties studied by Osamu Shimomura. 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. 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. 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. Frederick Tsuji's lab independently reported the expression of the recombinant protein one month later. 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. 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 is resistant to detergents, proteases, guanidinium chloride (GdmCl) treatments, and drastic temperature changes.
Due to the potential for widespread usage and the evolving needs of researchers, many different mutants of GFP have been engineered. The first major improvement was a single point mutation (S65T) reported in 1995 in Nature by Roger Tsien. 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 and Falkow. EGFP allowed the practical use of GFPs in mammalian cells. EGFP has an extinction coefficient (denoted ε) of 55,000 M−1cm−1. 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, a series of mutations that allow GFP to rapidly fold and mature even when fused to poorly folding peptides, was reported in 2006.
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 Zn biosensor.
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. 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. 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.
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.
The nomenclature of modified GFPs is often confusing due to overlapping mapping of several GFP versions onto a single name. For example, mGFP often refers to a GFP with an N-terminal palmitoylation that causes the GFP to bind to cell membranes. However, the same term is also used to refer to monomeric GFP, which is often achieved by the dimer interface breaking A206K mutation. Wild-type GFP has a weak dimerization tendency at concentrations above 5 mg/mL. mGFP also stands for "modified GFP," which has been optimized through amino acid exchange for stable expression in plant cells.
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.
Other fluorescent proteins
Because of the great variety of engineered GFP derivatives, fluorescent proteins that belong to a different family, such as the bilirubin-inducible fluorescent protein UnaG, dsRed, eqFP611, Dronpa, TagRFPs, KFP, EosFP, Dendra, IrisFP and many others, are erroneously referred to as GFP derivatives. Several of these proteins display unique properties like red-shifted emission above 600 nm or photoconversion from a green-emitting state to a red-emitting state. These properties are so far unique to fluorescent proteins other than GFP derivatives.
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.
A new class of fluorescent protein was evolved from a cyanobacterial (Trichodesmium erythraeum) phycobiliprotein, α-allophycocyanin, and named small ultra red fluorescent protein (smURFP) in 2016. smURFP autocatalytically self-incorporates the chromophore biliverdin without the need of an external protein, known as a lyase. Jellyfish- and coral-derived fluorescent proteins require oxygen and produce a stoichiometric amount of hydrogen peroxide upon chromophore formation. smURFP does not require oxygen or produce hydrogen peroxide and uses the chromophore, bliverdin. 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.
A review of new classes of fluorescent proteins and applications can be found in Trends in Biochemical Sciences.
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. 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. 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. The hydrogen-bonding network and electron-stacking interactions with these sidechains influence the color, intensity and photostability of GFP and its numerous derivatives. 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.
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 of the fact that it has not effect on the host once it is introduced to its cellular environment of the host. 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 were 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. 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.
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.
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. 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. 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.
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).
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). Genetically combining several spectral variants of GFP is a useful trick for the analysis of brain circuitry (Brainbow). Other interesting uses of fluorescent proteins in the literature include using FPs as sensors of neuron membrane potential, tracking of AMPA receptors on cell membranes, viral entry and the infection of individual influenza viruses and lentiviral viruses, etc.
It has also been found that new lines of transgenic GFP rats can be relevant for gene therapy as well as regenerative medicine. 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. Another application is the use of GFP co-transfection as internal control for transfection efficiency in mammalian cells.
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.
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.
Macro-scale biological processes, such as the spread of virus infections, can be followed using GFP labeling. In the past, mutagenic ultra violet light (UV) has been used to illuminate living organisms (e.g., see) to detect and photograph the GFP expression. Recently, a technique using non-mutagenic LED lights have been developed for macro-photography. The technique uses an epifluorescence camera attachment based on the same principle used in the construction of epifluorescence microscopes.
Alba, a green-fluorescent rabbit, was created by a French laboratory commissioned by Eduardo Kac using GFP for purposes of art and social commentary. 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. 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. A Japanese-American Team created green-fluorescent cats as proof of concept to use them potentially as model organisms for diseases, particularly HIV. 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.
Julian Voss-Andreae, a German-born artist specializing in "protein sculptures," created sculptures based on the structure of GFP, including the 1.70 m (5'6") tall "Green Fluorescent Protein" (2004) and the 1.40 m (4'7") 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.
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- Arun KH, Kaul CL, Ramarao P (2005). "Green fluorescent proteins in receptor research: an emerging tool for drug discovery". J Pharmacol Toxicol Methods. 51 (1): 1–23. doi:10.1016/j.vascn.2004.07.006. PMID 15596111.
- Song, Y. H.; Kim, C. S.; Seo, J. H. Noninvasive monitoring of environmental toxicity through green fluorescent protein expressing Escherichia coli. Korean J. Chem. Eng. 2016, 33 (4), 1331-1336.
- Pan, Y.; Leifert, A.; Graf, M.; Schiefer, F.; Thoröe-Boveleth, S.; Broda, J.; Halloran, M. C.; Hollert, H.; Laaf, D.; Simon, U.; Jahnen-Dechent, W. High-Sensitivity Real-Time Analysis of Nanoparticle Toxicity in Green Fluorescent Protein-Expressing Zebrafish. Small. 2013, 9 (6), 863-869.
- Chalfie M (Jun 2009). "GFP: Lighting up life". Proceedings of the National Academy of Sciences of the United States of America. 106 (25): 10073–10080. Bibcode:2009PNAS..10610073C. doi:10.1073/pnas.0904061106. PMC . PMID 19553219.
- Yuste R (Dec 2005). "Fluorescence microscopy today". Nature Methods. 2 (12): 902–4. doi:10.1038/nmeth1205-902. PMID 16299474.
- Komorowski M, Finkenstädt B, Rand D (Jun 2010). "Using a single fluorescent reporter gene to infer half-life of extrinsic noise and other parameters of gene expression". Biophysical Journal. 98 (12): 2759–2769. Bibcode:2010BpJ....98.2759K. doi:10.1016/j.bpj.2010.03.032. PMC . PMID 20550887.
- Gunkel M, Erdel F, Rippe K, Lemmer P, Kaufmann R, Hörmann C, Amberger R, Cremer C (Jun 2009). "Dual color localization microscopy of cellular nanostructures". Biotechnology Journal. 4 (6): 927–38. doi:10.1002/biot.200900005. PMID 19548231.
- Chudakov DM, Lukyanov S, Lukyanov KA (Dec 2005). "Fluorescent proteins as a toolkit for in vivo imaging". Trends in Biotechnology. 23 (12): 605–13. doi:10.1016/j.tibtech.2005.10.005. PMID 16269193.
- Livet J, Weissman TA, Kang H, Draft RW, Lu J, Bennis RA, Sanes JR, Lichtman JW (Nov 2007). "Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system". Nature. 450 (7166): 56–62. Bibcode:2007Natur.450...56L. doi:10.1038/nature06293. PMID 17972876.
- Baker BJ, Mutoh H, Dimitrov D, Akemann W, Perron A, Iwamoto Y, Jin L, Cohen LB, Isacoff EY, Pieribone VA, Hughes T, Knöpfel T (Aug 2008). "Genetically encoded fluorescent sensors of membrane potential". Brain Cell Biology. 36 (1–4): 53–67. doi:10.1007/s11068-008-9026-7. PMC . PMID 18679801.
- Adesnik H, Nicoll RA, England PM (Dec 2005). "Photoinactivation of native AMPA receptors reveals their real-time trafficking". Neuron. 48 (6): 977–85. doi:10.1016/j.neuron.2005.11.030. PMID 16364901.
- Lakadamyali M, Rust MJ, Babcock HP, Zhuang X (Aug 2003). "Visualizing infection of individual influenza viruses". Proceedings of the National Academy of Sciences of the United States of America. 100 (16): 9280–5. Bibcode:2003PNAS..100.9280L. doi:10.1073/pnas.0832269100. PMC . PMID 12883000.
- Joo KI, Wang P (Oct 2008). "Visualization of targeted transduction by engineered lentiviral vectors". Gene Therapy. 15 (20): 1384–96. doi:10.1038/gt.2008.87. PMC . PMID 18480844.
- 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 (Oct 2010). "New lines of GFP transgenic rats relevant for regenerative medicine and gene therapy". Transgenic Research. 19 (5): 745–63. doi:10.1007/s11248-009-9352-2. PMID 20094912.
- Elliott G, McGrath J, Crockett-Torabi E (Jun 2000). "Green fluorescent protein: A novel viability assay for cryobiological applications". Cryobiology. 40 (4): 360–369. doi:10.1006/cryo.2000.2258. PMID 10924267.
- Fakhrudin N, Ladurner A, Atanasov AG, Heiss EH, Baumgartner L, Markt P, Schuster D, Ellmerer EP, Wolber G, Rollinger JM, Stuppner H, Dirsch VM (Apr 2010). "Computer-aided discovery, validation, and mechanistic characterization of novel neolignan activators of peroxisome proliferator-activated receptor gamma". Molecular Pharmacology. 77 (4): 559–66. doi:10.1124/mol.109.062141. PMC . PMID 20064974.
- Gather MC, Yun SH (2011). "Single-cell biological lasers". Nature Photonics. 5 (7): 406–410. Bibcode:2011NaPho...5..406G. doi:10.1038/nphoton.2011.99.
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- Kouros-Mehr H, Bechis SK, Slorach EM, Littlepage LE, Egeblad M, Ewald AJ, Pai SY, Ho IC, Werb Z (Feb 2008). "GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model". Cancer Cell. 13 (2): 141–52. doi:10.1016/j.ccr.2008.01.011. PMC . PMID 18242514.
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- Zhu YJ, Agbayani R, Moore PH (Apr 2004). "Green fluorescent protein as a visual selection marker for papaya (Carica papaya L.) transformation". Plant Cell Reports. 22 (9): 660–7. doi:10.1007/s00299-004-0755-5. PMID 14749892.
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- "PlantEdDL - Using SRL digital cameras in quantitative investigations of plants expressing green fluorescent protein (GFP)". planted.botany.org. Retrieved 2016-03-23.
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|Library resources about
Green fluorescent protein
|Wikimedia Commons has media related to Green fluorescent proteins.|
- A comprehensive article on fluorescent proteins at Scholarpedia
- Brief summary of landmark GFP papers
- Interactive Java applet demonstrating the chemistry behind the formation of the GFP chromophore
- Video of 2008 Nobel Prize lecture of Roger Tsien on fluorescent proteins
- Excitation and emission spectra for various fluorescent proteins
- 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
- Molecule of the Month, June 2003: an illustrated overview of GFP by David Goodsell.
- Molecule of the Month, June 2014: an illustrated overview of GFP-like variants by David Goodsell.
This tab holds the annotation information that is stored in the Pfam database. As we move to using Wikipedia as our main source of annotation, the contents of this tab will be gradually replaced by the Wikipedia tab.
Green fluorescent protein Provide feedback
No Pfam abstract.
External database links
This tab holds annotation information from the InterPro database.
InterPro entry IPR011584
The green fluorescent protein (GFP) is found in the jellyfish (Aequorea victoria), and functions as an energy-transfer acceptor. It fluoresces in vivo upon receiving energy from the Ca2+-activated photoprotein aequorin. The protein absorbs light maximally at 395 nm and exhibits a smaller absorbance peak at 470 nm. The fluorescence emission spectrum peaks at 509 nm with a shoulder at 540 nm. The protein is produced in the photocytes and contains a chromophore, which is composed of modified amino acid residues. The chromophore is formed upon cyclisation of the residues ser-dehydrotyr-gly. There are several other members of the GFP family, which are able to fluoresce different colours, several of which are non-fluorescent [PUBMED:10852900]. These proteins are all essentially encoded by single genes, since both the substrate and the catalytic enzyme for pigment biosynthesis are provided within a single polypeptide chain [PUBMED:12325128].
The mapping between Pfam and Gene Ontology is provided by InterPro. If you use this data please cite InterPro.
|Biological process||bioluminescence (GO:0008218)|
Below is a listing of the unique domain organisations or architectures in which this domain is found. More...
The graphic that is shown by default represents the longest sequence with a given architecture. Each row contains the following information:
- the number of sequences which exhibit this architecture
a textual description of the architecture, e.g. Gla, EGF x 2, Trypsin.
This example describes an architecture with one
Gladomain, followed by two consecutive
EGFdomains, and finally a single
- a link to the page in the Pfam site showing information about the sequence that the graphic describes
- the UniProt description of the protein sequence
- the number of residues in the sequence
- the Pfam graphic itself.
Note that you can see the family page for a particular domain by clicking on the graphic. You can also choose to see all sequences which have a given architecture by clicking on the Show link in each row.
Finally, because some families can be found in a very large number of architectures, we load only the first fifty architectures by default. If you want to see more architectures, click the button at the bottom of the page to load the next set.
Loading domain graphics...
This superfamily has an unusual fold of an 11 stranded beta barrel enclosing an alpha-helix. This superfamily includes green fluorescent protein as well as a domain from nidogen.
The clan contains the following 2 members:G2F GFP
We store a range of different sequence alignments for families. As well as the seed alignment from which the family is built, we provide the full alignment, generated by searching the sequence database (reference proteomes) using the family HMM. We also generate alignments using four representative proteomes (RP) sets, the UniProtKB sequence database, the NCBI sequence database, and our metagenomics sequence database. More...
There are various ways to view or download the sequence alignments that we store. We provide several sequence viewers and a plain-text Stockholm-format file for download.
We make a range of alignments for each Pfam-A family:
- the curated alignment from which the HMM for the family is built
- the alignment generated by searching the sequence database using the HMM
- Representative Proteomes (RPs) at 15%, 35%, 55% and 75% co-membership thresholds
- alignment generated by searching the UniProtKB sequence database using the family HMM
- alignment generated by searching the NCBI sequence database using the family HMM
- alignment generated by searching the metagenomics sequence database using the family HMM
You can see the alignments as HTML or in three different sequence viewers:
- a Java applet developed at the University of Dundee. You will need Java installed before running jalview
- an HTML page showing the whole alignment.Please note: full Pfam alignments can be very large. These HTML views are extremely large and often cause problems for browsers. Please use either jalview or the Pfam viewer if you have trouble viewing the HTML version
- an HTML-based representation of the alignment, coloured according to the posterior-probability (PP) values from the HMM. As for the standard HTML view, heatmap alignments can also be very large and slow to render.
You can download (or view in your browser) a text representation of a Pfam alignment in various formats:
You can also change the order in which sequences are listed in the alignment, change how insertions are represented, alter the characters that are used to represent gaps in sequences and, finally, choose whether to download the alignment or to view it in your browser directly.
You may find that large alignments cause problems for the viewers and the reformatting tool, so we also provide all alignments in Stockholm format. You can download either the plain text alignment, or a gzipped version of it.
We make a range of alignments for each Pfam-A family. You can see a description of each above. You can view these alignments in various ways but please note that some types of alignment are never generated while others may not be available for all families, most commonly because the alignments are too large to handle.
1Cannot generate PP/Heatmap alignments for seeds; no PP data available
Key: available, not generated, — not available.
Format an alignment
We make all of our alignments available in Stockholm format. You can download them here as raw, plain text files or as gzip-compressed files.
You can also download a FASTA format file containing the full-length sequences for all sequences in the full alignment.
HMM logos is one way of visualising profile HMMs. Logos provide a quick overview of the properties of an HMM in a graphical form. You can see a more detailed description of HMM logos and find out how you can interpret them here. More...
If you find these logos useful in your own work, please consider citing the following article:
This page displays the phylogenetic tree for this family's seed alignment. We use FastTree to calculate neighbour join trees with a local bootstrap based on 100 resamples (shown next to the tree nodes). FastTree calculates approximately-maximum-likelihood phylogenetic trees from our seed alignment.
Note: You can also download the data file for the tree.
Curation and family details
This section shows the detailed information about the Pfam family. You can see the definitions of many of the terms in this section in the glossary and a fuller explanation of the scoring system that we use in the scores section of the help pages.
|Number in seed:||3|
|Number in full:||26|
|Average length of the domain:||186.20 aa|
|Average identity of full alignment:||34 %|
|Average coverage of the sequence by the domain:||81.01 %|
|HMM build commands:||
build method: hmmbuild -o /dev/null HMM SEED
search method: hmmsearch -Z 45638612 -E 1000 --cpu 4 HMM pfamseq
|Family (HMM) version:||22|
|Download:||download the raw HMM for this family|
Weight segments by...
Change the size of the sunburst
selected sequences to HMM
a FASTA-format file
- 0 sequences
- 0 species
This visualisation provides a simple graphical representation of the distribution of this family across species. You can find the original interactive tree in the More....
This chart is a modified "sunburst" visualisation of the species tree for this family. It shows each node in the tree as a separate arc, arranged radially with the superkingdoms at the centre and the species arrayed around the outermost ring.
How the sunburst is generated
The tree is built by considering the taxonomic lineage of each sequence that has a match to this family. For each node in the resulting tree, we draw an arc in the sunburst. The radius of the arc, its distance from the root node at the centre of the sunburst, shows the taxonomic level ("superkingdom", "kingdom", etc). The length of the arc represents either the number of sequences represented at a given level, or the number of species that are found beneath the node in the tree. The weighting scheme can be changed using the sunburst controls.
In order to reduce the complexity of the representation, we reduce the number of taxonomic levels that we show. We consider only the following eight major taxonomic levels:
Colouring and labels
Segments of the tree are coloured approximately according to their superkingdom. For example, archeal branches are coloured with shades of orange, eukaryotes in shades of purple, etc. The colour assignments are shown under the sunburst controls. Where space allows, the name of the taxonomic level will be written on the arc itself.
As you move your mouse across the sunburst, the current node will be highlighted. In the top section of the controls panel we show a summary of the lineage of the currently highlighed node. If you pause over an arc, a tooltip will be shown, giving the name of the taxonomic level in the title and a summary of the number of sequences and species below that node in the tree.
Anomalies in the taxonomy tree
There are some situations that the sunburst tree cannot easily handle and for which we have work-arounds in place.
Missing taxonomic levels
Some species in the taxonomic tree may not have one or more of the main eight levels that we display. For example, Bos taurus is not assigned an order in the NCBI taxonomic tree. In such cases we mark the omitted level with, for example, "No order", in both the tooltip and the lineage summary.
Unmapped species names
The tree is built by looking at each sequence in the full alignment for the family. We take the name of the species given by UniProt and try to map that to the full taxonomic tree from NCBI. In some cases, the name chosen by UniProt does not map to any node in the NCBI tree, perhaps because the chosen name is listed as a synonym or a misspelling in the NCBI taxonomy.
So that these nodes are not simply omitted from the sunburst tree, we group them together in a separate branch (or segment of the sunburst tree). Since we cannot determine the lineage for these unmapped species, we show all levels between the superkingdom and the species as "uncategorised".
Since we reduce the species tree to only the eight main taxonomic levels, sequences that are mapped to the sub-species level in the tree would not normally be shown. Rather than leave out these species, we map them instead to their parent species. So, for example, for sequences belonging to one of the Vibrio cholerae sub-species in the NCBI taxonomy, we show them instead as belonging to the species Vibrio cholerae.
Too many species/sequences
For large species trees, you may see blank regions in the outer layers of the sunburst. These occur when there are large numbers of arcs to be drawn in a small space. If an arc is less than approximately one pixel wide, it will not be drawn and the space will be left blank. You may still be able to get some information about the species in that region by moving your mouse across the area, but since each arc will be very small, it will be difficult to accurately locate a particular species.
The tree shows the occurrence of this domain across different species. More...
We show the species tree in one of two ways. For smaller trees we try to show an interactive representation, which allows you to select specific nodes in the tree and view them as an alignment or as a set of Pfam domain graphics.
Unfortunately we have found that there are problems viewing the interactive tree when the it becomes larger than a certain limit. Furthermore, we have found that Internet Explorer can become unresponsive when viewing some trees, regardless of their size. We therefore show a text representation of the species tree when the size is above a certain limit or if you are using Internet Explorer to view the site.
If you are using IE you can still load the interactive tree by clicking the "Generate interactive tree" button, but please be aware of the potential problems that the interactive species tree can cause.
For all of the domain matches in a full alignment, we count the number that are found on all sequences in the alignment. This total is shown in the purple box.
We also count the number of unique sequences on which each domain is found, which is shown in green. Note that a domain may appear multiple times on the same sequence, leading to the difference between these two numbers.
Finally, we group sequences from the same organism according to the NCBI code that is assigned by UniProt, allowing us to count the number of distinct sequences on which the domain is found. This value is shown in the pink boxes.
We use the NCBI species tree to group organisms according to their taxonomy and this forms the structure of the displayed tree. Note that in some cases the trees are too large (have too many nodes) to allow us to build an interactive tree, but in most cases you can still view the tree in a plain text, non-interactive representation. Those species which are represented in the seed alignment for this domain are highlighted.
You can use the tree controls to manipulate how the interactive tree is displayed:
- show/hide the summary boxes
- highlight species that are represented in the seed alignment
- expand/collapse the tree or expand it to a given depth
- select a sub-tree or a set of species within the tree and view them graphically or as an alignment
- save a plain text representation of the tree
Please note: for large trees this can take some time. While the tree is loading, you can safely switch away from this tab but if you browse away from the family page entirely, the tree will not be loaded.
There are 5 interactions for this family. More...
We determine these interactions using iPfam, which considers the interactions between residues in three-dimensional protein structures and maps those interactions back to Pfam families. You can find more information about the iPfam algorithm in the journal article that accompanies the website.
For those sequences which have a structure in the Protein DataBank, we use the mapping between UniProt, PDB and Pfam coordinate systems from the PDBe group, to allow us to map Pfam domains onto UniProt sequences and three-dimensional protein structures. The table below shows the structures on which the GFP domain has been found. There are 1212 instances of this domain found in the PDB. Note that there may be multiple copies of the domain in a single PDB structure, since many structures contain multiple copies of the same protein sequence.
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