The green fluorescent protein (GFP) is a
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
that exhibits bright green
fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
when exposed to light in the blue to
ultraviolet
Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nanometer, nm (with a corresponding frequency around 30 Hertz, PHz) to 400 nm (750 Hertz, THz), shorter than that of visible light, but longer than ...
range.
The label ''GFP'' traditionally refers to the protein first isolated from the
jellyfish
Jellyfish and sea jellies are the informal common names given to the medusa-phase of certain gelatinous members of the subphylum Medusozoa, a major part of the phylum Cnidaria. Jellyfish are mainly free-swimming marine animals with umbrella- ...
''
Aequorea victoria
''Aequorea victoria'', also sometimes called the crystal jelly, is a bioluminescent hydrozoan jellyfish, or hydromedusa, that is found off the west coast of North America.
The species is best known as the source of two proteins involved in biolu ...
'' and is sometimes called ''avGFP''. However, GFPs have been found in other organisms including
coral
Corals are marine invertebrates within the class Anthozoa of the phylum Cnidaria. They typically form compact colonies of many identical individual polyps. Coral species include the important reef builders that inhabit tropical oceans and sec ...
s,
sea anemone
Sea anemones are a group of predation, predatory marine invertebrates of the order (biology), order Actiniaria. Because of their colourful appearance, they are named after the ''Anemone'', a terrestrial flowering plant. Sea anemones are classifi ...
s,
zoanithids,
copepod
Copepods (; meaning "oar-feet") are a group of small crustaceans found in nearly every freshwater and saltwater habitat (ecology), habitat. Some species are planktonic (inhabiting sea waters), some are benthos, benthic (living on the ocean floor) ...
s and
lancelet
The lancelets ( or ), also known as amphioxi (singular: amphioxus ), consist of some 30 to 35 species of "fish-like" benthic filter feeding chordates in the order Amphioxiformes. They are the modern representatives of the subphylum Cephalochorda ...
s.
The GFP from ''A. victoria'' has a major
excitation peak at a
wavelength
In physics, the wavelength is the spatial period of a periodic wave—the distance over which the wave's shape repeats.
It is the distance between consecutive corresponding points of the same phase on the wave, such as two adjacent crests, tro ...
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 visible spectrum is the portion of the electromagnetic spectrum that is visual perception, visible to the human eye. Electromagnetic radiation in this range of wavelengths is called ''visible light'' or simply light. A typical human eye wil ...
. The fluorescence
quantum yield The quantum yield (Φ) of a radiation-induced process is the number of times a specific event occurs per photon absorbed by the system.
Applications
Fluorescence spectroscopy
The fluorescence quantum yield is defined as the ratio of the numb ...
(QY) of GFP is 0.79. The GFP from the
sea pansy
The sea pansy, ''Renilla reniformis,'' is a species of colonial cnidarian in the family Renillidae, part of an octocoral subclass of Anthozoa that inhabit an expansive range of environments. It is native to warm continental shelf waters of th ...
(''
Renilla reniformis
The sea pansy, ''Renilla reniformis,'' is a species of colonial cnidarian in the family Renillidae, part of an octocoral subclass of Anthozoa that inhabit an expansive range of environments. It is native to warm continental shelf waters of th ...
'') 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 an internal
chromophore
A chromophore is the part of a molecule responsible for its color.
The color that is seen by our eyes is the one not absorbed by the reflecting object within a certain wavelength spectrum of visible light. The chromophore is a region in the molec ...
without requiring any accessory
cofactors, gene products, or
enzymes
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different molecule ...
/
substrates other than molecular oxygen.
In
cell
Cell most often refers to:
* Cell (biology), the functional basic unit of life
Cell may also refer to:
Locations
* Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery ...
and
molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
, the GFP
gene
In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
is frequently used as a
reporter of expression.
It has been used in modified forms to make
biosensor
A biosensor is an analytical device, used for the detection of a chemical substance, that combines a biological component with a physicochemical detector.
The ''sensitive biological element'', e.g. tissue, microorganisms, organelles, cell recep ...
s, and many animals have been created that express GFP, which demonstrates a
proof of concept
Proof of concept (POC or PoC), also known as proof of principle, is a realization of a certain method or idea in order to demonstrate its feasibility, or a demonstration in principle with the aim of verifying that some concept or theory has prac ...
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
was a Japanese organic chemist and marine biologist, and Professor Emeritus at Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts and Boston University School of Medicine. He was awarded the Nobel Prize in Chemistry in 2008 for the ...
, and
Martin Chalfie
Martin Lee Chalfie (born January 15, 1947) is an American scientist. He is University Professor at Columbia University. He shared the 2008 Nobel Prize in Chemistry along with Osamu Shimomura and Roger Y. Tsien "for the discovery and development ...
were awarded the 2008
Nobel Prize in Chemistry
)
, image = Nobel Prize.png
, alt = A golden medallion with an embossed image of a bearded man facing left in profile. To the left of the man is the text "ALFR•" then "NOBEL", and on the right, the text (smaller) "NAT•" then "M ...
on 10 October 2008 for their discovery and development of the green fluorescent protein.
Most commercially available genes for GFP and similar fluorescent proteins are around 730 base-pairs long. The natural protein has 238 amino acids. Its molecular mass is 27 kD.
Therefore, fusing the GFP gene to the gene of a protein of interest can significantly increase the protein's size and molecular mass, and can impair the protein's natural function or change its location or trajectory of transport within the cell.
Background
Wild-type GFP (wtGFP)
In the 1960s and 1970s, GFP, along with the separate luminescent protein
aequorin
Aequorin is a calcium-activated photoprotein isolated from the hydrozoan ''Aequorea victoria''. Its bioluminescence was studied decades before the protein was isolated from the animal by Osamu Shimomura in 1962. In the animal, the protein occurs ...
(an
enzyme
Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. A ...
that catalyzes the breakdown of
luciferin
Luciferin (from the Latin ''lucifer'', "light-bearer") is a generic term for the light-emitting compound found in organisms that generate bioluminescence. Luciferins typically undergo an enzyme-catalyzed reaction with molecular oxygen. The result ...
, releasing light), was first purified from the jellyfish ''Aequorea victoria'' and its properties studied by
Osamu Shimomura
was a Japanese organic chemist and marine biologist, and Professor Emeritus at Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts and Boston University School of Medicine. He was awarded the Nobel Prize in Chemistry in 2008 for the ...
.
In ''A. victoria'', GFP fluorescence occurs when
aequorin
Aequorin is a calcium-activated photoprotein isolated from the hydrozoan ''Aequorea victoria''. Its bioluminescence was studied decades before the protein was isolated from the animal by Osamu Shimomura in 1962. In the animal, the protein occurs ...
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
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...
samples to several labs. The lab of
Martin Chalfie
Martin Lee Chalfie (born January 15, 1947) is an American scientist. He is University Professor at Columbia University. He shared the 2008 Nobel Prize in Chemistry along with Osamu Shimomura and Roger Y. Tsien "for the discovery and development ...
expressed the coding sequence of wtGFP, with the first few amino acids deleted, in heterologous cells of ''
E. coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
'' and ''
C. elegans
''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a blend of the Greek ''caeno-'' (recent), ''rhabditis'' (r ...
'', 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
A chromophore is the part of a molecule responsible for its color.
The color that is seen by our eyes is the one not absorbed by the reflecting object within a certain wavelength spectrum of visible light. The chromophore is a region in the molec ...
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.
GFP derivatives
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
Roger Yonchien Tsien (pronounced , "'' CHEN''"'';'' February 1, 1952 – August 24, 2016) was an American biochemist. He was a professor of chemistry and biochemistry at the University of California, San Diego and was awarded the Nobel Prize in ...
.
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 The quantum yield (Φ) of a radiation-induced process is the number of times a specific event occurs per photon absorbed by the system.
Applications
Fluorescence spectroscopy
The fluorescence quantum yield is defined as the ratio of the numb ...
(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.
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
Yellow fluorescent protein (YFP) is a genetic mutant of green fluorescent protein (GFP) originally derived from the jellyfish ''Aequorea victoria''. Its excitation peak is 513 nm and its emission peak is 527 nm. Like the parent GFP, YFP ...
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.
Chromophore binding. The critical mutation in cyan derivatives is the Y66W substitution, which causes the chromophore to form with an
indole
Indole is an aromatic heterocyclic organic compound with the formula C8 H7 N. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered pyrrole ring. Indole is widely distributed in the natural environmen ...
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
Förster resonance energy transfer (FRET), fluorescence resonance energy transfer, resonance energy transfer (RET) or electronic energy transfer (EET) is a mechanism describing energy transfer between two light-sensitive molecules ( chromophores). ...
(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
Synaptobrevins (''synaptobrevin isotypes 1-2'') are small integral membrane proteins of secretory vesicles with molecular weight of 18 kilodalton (kDa) that are part of the vesicle-associated membrane protein (VAMP) family.
Synaptobrevi ...
have been used to visualize synaptic activity in neurons.
Redox sensitive GFP (
roGFP
The reduction-oxidation sensitive green fluorescent protein (roGFP) is a green fluorescent protein engineered to be sensitive to changes in the local redox environment. roGFPs are used as redox-sensitive biosensors.
In 2004, researchers in S. J ...
) was engineered by introduction of cysteines into the beta barrel structure. The
redox
Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate (chemistry), substrate change. Oxidation is the loss of Electron, electrons or an increase in the oxidation state, while reduction ...
state of the cysteines determines the
fluorescent
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
properties of
roGFP
The reduction-oxidation sensitive green fluorescent protein (roGFP) is a green fluorescent protein engineered to be sensitive to changes in the local redox environment. roGFPs are used as redox-sensitive biosensors.
In 2004, researchers in S. J ...
.
Nomenclature
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
Palmitoylation is the covalent attachment of fatty acids, such as palmitic acid, to cysteine (''S''-palmitoylation) and less frequently to serine and threonine (''O''-palmitoylation) residues of proteins, which are typically lipid bilayer, memb ...
that causes the GFP to bind to
cell membrane
The cell membrane (also known as the plasma membrane (PM) or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of all cells from the outside environment ( ...
s. However, the same term is also used to refer to
monomer
In chemistry, a monomer ( ; ''mono-'', "one" + '' -mer'', "part") is a molecule that can react together with other monomer molecules to form a larger polymer chain or three-dimensional network in a process called polymerization.
Classification
Mo ...
ic GFP, which is often achieved by the dimer interface breaking A206K mutation.
Wild-type GFP has a weak
dimer
Dimer may refer to:
* Dimer (chemistry), a chemical structure formed from two similar sub-units
** Protein dimer, a protein quaternary structure
** d-dimer
* Dimer model, an item in statistical mechanics, based on ''domino tiling''
* Julius Dimer ...
ization 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.
In nature
![Pone](https://upload.wikimedia.org/wikipedia/commons/6/61/Pone.0011517.g001.png)
The purpose of both the (primary)
bioluminescence
Bioluminescence is the production and emission of light by living organisms. It is a form of chemiluminescence. Bioluminescence occurs widely in marine vertebrates and invertebrates, as well as in some fungi, microorganisms including some b ...
(from
aequorin
Aequorin is a calcium-activated photoprotein isolated from the hydrozoan ''Aequorea victoria''. Its bioluminescence was studied decades before the protein was isolated from the animal by Osamu Shimomura in 1962. In the animal, the protein occurs ...
's action on luciferin) and the (secondary)
fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation. It is a form of luminescence. In most cases, the emitted light has a longer wavelength, and therefore a lower photon energy, tha ...
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
A chromophore is the part of a molecule responsible for its color.
The color that is seen by our eyes is the one not absorbed by the reflecting object within a certain wavelength spectrum of visible light. The chromophore is a region in the molec ...
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
The lancelets ( or ), also known as amphioxi (singular: amphioxus ), consist of some 30 to 35 species of "fish-like" benthic filter feeding chordates in the order Amphioxiformes. They are the modern representatives of the subphylum Cephalochorda ...
are known to produce GFP in various regions of their body.
Unlike ''
A. victoria'', lancelets do not produce their own blue light, and the origin of their
endogenous
Endogenous substances and processes are those that originate from within a living system such as an organism, tissue, or cell.
In contrast, exogenous substances and processes are those that originate from outside of an organism.
For example, es ...
GFP is still unknown. Some speculate that it attracts
plankton
Plankton are the diverse collection of organisms found in Hydrosphere, water (or atmosphere, air) that are unable to propel themselves against a Ocean current, current (or wind). The individual organisms constituting plankton are called plankt ...
towards the mouth of the lancelet, serving as a passive hunting mechanism. It may also serve as a
photoprotective
Photoprotection is the biochemical process that helps organisms cope with molecular damage caused by sunlight. Plants and other oxygenic phototrophs have developed a suite of photoprotective mechanisms to prevent photoinhibition and oxidative st ...
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
Marine is an adjective meaning of or pertaining to the sea or ocean.
Marine or marines may refer to:
Ocean
* Maritime (disambiguation)
* Marine art
* Marine biology
* Marine debris
* Marine habitats
* Marine life
* Marine pollution
Military
* ...
copepod
Copepods (; meaning "oar-feet") are a group of small crustaceans found in nearly every freshwater and saltwater habitat (ecology), habitat. Some species are planktonic (inhabiting sea waters), some are benthos, benthic (living on the ocean floor) ...
s, particularly from the
Pontellidae
Pontellidae is a family of copepods in the order Calanoida, containing the following genera:
*'' Anomalocera'' Templeton, 1837
*'' Calanopia'' Dana, 1852
*'' Epilabidocera'' C. B. Wilson, 1932
*'' Isocope'' Brady, 1915
*'' Ivellopsis'' Claus, 189 ...
and
Aetideidae
Aetideidae is a family of copepods belonging to the order Calanoida.
Genera
Genera:
* ''Aetideopsis'' Sars, 1903
* ''Aetideus'' Brady, 1883
* ''Aetidius'' Brady, 1883
References
{{Taxonbar, from=Q3606128
Copepods ...
families.
GFP isolated from ''
Pontella mimocerami
''Pontella'' is a marine copepod genus in the family Pontellidae. It is an organism that bears three lenses in the eye. The outer has a parabola, parabolic surface, countering the effects of spherical aberration while allowing a sharp image to be ...
'' has shown high levels of brightness with a
quantum yield The quantum yield (Φ) of a radiation-induced process is the number of times a specific event occurs per photon absorbed by the system.
Applications
Fluorescence spectroscopy
The fluorescence quantum yield is defined as the ratio of the numb ...
of 0.92, making them nearly two-fold brighter than the commonly used EGFP isolated from ''A. victoria.''
Other fluorescent proteins
![Fluorescence from Fluorescent Proteins](https://upload.wikimedia.org/wikipedia/commons/c/c3/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. When GFP-like barrels of different spectra characteristics are used, the excitation spectra of one chromophore can be used to power another chromophore (FRET), allowing for conversion between wavelengths of light.
FMN-binding fluorescent proteins
A FMN-binding fluorescent protein (FbFP), also known as a LOV-based fluorescent protein, is a small, oxygen-independent fluorescent protein that binds flavin mononucleotide (FMN) as a chromophore.
They were developed from blue-light receptors (so ...
(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.
![White Light Image of Fluorescent Proteins](https://upload.wikimedia.org/wikipedia/commons/0/00/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 Fluorescent proteins include:
* Green fluorescent protein (GFP)
* Yellow fluorescent protein
Yellow fluorescent protein (YFP) is a genetic mutant of green fluorescent protein (GFP) originally derived from the jellyfish '' Aequorea victoria''. Its ...
was evolved from a
cyanobacteria
Cyanobacteria (), also known as Cyanophyta, are a phylum of gram-negative bacteria that obtain energy via photosynthesis. The name ''cyanobacteria'' refers to their color (), which similarly forms the basis of cyanobacteria's common name, blu ...
l (''
Trichodesmium erythraeum'')
phycobiliprotein
Phycobiliproteins are water-soluble proteins present in cyanobacteria and certain algae (rhodophytes, cryptomonads, glaucocystophytes). They capture light energy, which is then passed on to chlorophylls during photosynthesis. Phycobiliproteins are ...
, α-
allophycocyanin
Allophycocyanin ("other algal blue protein"; from Greek: '' (allos)'' meaning "other", '' (phykos)'' meaning “alga”, and '' (kyanos)'' meaning "blue") is a protein from the light-harvesting phycobiliprotein family, along with phycocyanin, phyco ...
, and named small ultra
red fluorescent protein
Red fluorescent protein (RFP) is a fluorophore that fluoresces red-orange when excited. Several variants have been developed using directed mutagenesis. The original was isolated from Discosoma, and named DsRed. Others are now available that flu ...
(
smURFP) in 2016.
smURFP autocatalytically self-incorporates the
chromophore
A chromophore is the part of a molecule responsible for its color.
The color that is seen by our eyes is the one not absorbed by the reflecting object within a certain wavelength spectrum of visible light. The chromophore is a region in the molec ...
biliverdin
Biliverdin (latin for green bile) is a green tetrapyrrolic bile pigment, and is a product of heme catabolism.Boron W, Boulpaep E. Medical Physiology: a cellular and molecular approach, 2005. 984-986. Elsevier Saunders, United States. It is the pi ...
without the need of an external
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
, known as a
lyase
In biochemistry, a lyase is an enzyme that catalyzes the breaking (an elimination reaction) of various chemical bonds by means other than hydrolysis (a substitution reaction) and oxidation, often forming a new double bond or a new ring structure. ...
.
Jellyfish
Jellyfish and sea jellies are the informal common names given to the medusa-phase of certain gelatinous members of the subphylum Medusozoa, a major part of the phylum Cnidaria. Jellyfish are mainly free-swimming marine animals with umbrella- ...
- and
coral
Corals are marine invertebrates within the class Anthozoa of the phylum Cnidaria. They typically form compact colonies of many identical individual polyps. Coral species include the important reef builders that inhabit tropical oceans and sec ...
-derived GFP-like proteins require
oxygen
Oxygen is the chemical element with the symbol O and atomic number 8. It is a member of the chalcogen group in the periodic table, a highly reactive nonmetal, and an oxidizing agent that readily forms oxides with most elements as wel ...
and produce a
stoichiometric
Stoichiometry refers to the relationship between the quantities of reactants and products before, during, and following chemical reactions.
Stoichiometry is founded on the law of conservation of mass where the total mass of the reactants equal ...
amount of
hydrogen peroxide
Hydrogen peroxide is a chemical compound with the formula . In its pure form, it is a very pale blue liquid that is slightly more viscous than water. It is used as an oxidizer, bleaching agent, and antiseptic, usually as a dilute solution (3% ...
upon
chromophore
A chromophore is the part of a molecule responsible for its color.
The color that is seen by our eyes is the one not absorbed by the reflecting object within a certain wavelength spectrum of visible light. The chromophore is a region in the molec ...
formation.
smURFP does not require
oxygen
Oxygen is the chemical element with the symbol O and atomic number 8. It is a member of the chalcogen group in the periodic table, a highly reactive nonmetal, and an oxidizing agent that readily forms oxides with most elements as wel ...
or produce
hydrogen peroxide
Hydrogen peroxide is a chemical compound with the formula . In its pure form, it is a very pale blue liquid that is slightly more viscous than water. It is used as an oxidizer, bleaching agent, and antiseptic, usually as a dilute solution (3% ...
and uses the
chromophore
A chromophore is the part of a molecule responsible for its color.
The color that is seen by our eyes is the one not absorbed by the reflecting object within a certain wavelength spectrum of visible light. The chromophore is a region in the molec ...
,
biliverdin
Biliverdin (latin for green bile) is a green tetrapyrrolic bile pigment, and is a product of heme catabolism.Boron W, Boulpaep E. Medical Physiology: a cellular and molecular approach, 2005. 984-986. Elsevier Saunders, United States. It is the pi ...
.
smURFP has a large
extinction coefficient (180,000 M
−1 cm
−1) and has a modest
quantum yield The quantum yield (Φ) of a radiation-induced process is the number of times a specific event occurs per photon absorbed by the system.
Applications
Fluorescence spectroscopy
The fluorescence quantum yield is defined as the ratio of the numb ...
(0.20), which makes it comparable biophysical brightness to
eGFP
The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea ...
and ~2-fold brighter than most red or far-red
fluorescent protein Fluorescent proteins include:
* Green fluorescent protein (GFP)
* Yellow fluorescent protein
Yellow fluorescent protein (YFP) is a genetic mutant of green fluorescent protein (GFP) originally derived from the jellyfish '' Aequorea victoria''. Its ...
s derived from
coral
Corals are marine invertebrates within the class Anthozoa of the phylum Cnidaria. They typically form compact colonies of many identical individual polyps. Coral species include the important reef builders that inhabit tropical oceans and sec ...
.
smURFP spectral properties are similar to the organic dye
Cy5
Cyanines, also referred to as tetramethylindo(di)-carbocyanines are a synthetic dye family belonging to the polymethine group. Although the name derives etymologically from terms for shades of blue, the cyanine family covers the electromagnetic s ...
.
![E](https://upload.wikimedia.org/wikipedia/commons/4/49/E._coli_expressing_fluorescent_proteins.jpg)
Reviews on new classes of fluorescent proteins and applications can be found in the cited reviews.
Structure
GFP has a
beta barrel
In protein structures, a beta barrel is a beta sheet composed of tandem repeats that twists and coils to form a closed toroidal structure in which the first strand is bonded to the last strand (hydrogen bond). Beta-strands in many beta-barrels are ...
structure consisting of eleven β-strands with a pleated sheet arrangement, with an alpha helix containing the covalently bonded
chromophore
A chromophore is the part of a molecule responsible for its color.
The color that is seen by our eyes is the one not absorbed by the reflecting object within a certain wavelength spectrum of visible light. The chromophore is a region in the molec ...
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
In protein structures, a beta barrel is a beta sheet composed of tandem repeats that twists and coils to form a closed toroidal structure in which the first strand is bonded to the last strand (hydrogen bond). Beta-strands in many beta-barrels are ...
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
A chromophore is the part of a molecule responsible for its color.
The color that is seen by our eyes is the one not absorbed by the reflecting object within a certain wavelength spectrum of visible light. The chromophore is a region in the molec ...
formation. This process of
post-translational modification
Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribosome ...
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
A chromophore is the part of a molecule responsible for its color.
The color that is seen by our eyes is the one not absorbed by the reflecting object within a certain wavelength spectrum of visible light. The chromophore is a region in the molec ...
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.
Autocatalytic formation of the chromophore in wtGFP
![GFP mechanism](https://upload.wikimedia.org/wikipedia/commons/0/08/GFP_mechanism.svg)
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.
The formed HBI fluorophore is highlighted in green.
The reactions are catalyzed by residues Glu222 and Arg96.
An analogous mechanism is also possible with threonine in place of Ser65.
Applications
Reporter assays
Green fluorescent protein may be used as a
reporter gene
In molecular biology, a reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in bacteria, cell culture, animals or plants. Such genes are called reporters because the charac ...
.
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. 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.
Advantages
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.
Fluorescence microscopy
The availability of GFP and its derivatives has thoroughly redefined
fluorescence microscopy
A fluorescence microscope is an optical microscope that uses fluorescence instead of, or in addition to, scattering, reflection, and attenuation or absorption, to study the properties of organic or inorganic substances. "Fluorescence microsc ...
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
Phototoxicity, also called photoirritation, is a chemically induced skin irritation, requiring light, that does not involve the immune system. It is a type of photosensitivity.
The skin response resembles an exaggerated sunburn. The involved chemi ...
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. In yet another example, scientist 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. 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
For example, GFP had been widely used in labelling the
spermatozoa
A spermatozoon (; also spelled spermatozoön; ; ) is a motile sperm cell, or moving form of the haploid cell that is the male gamete. A spermatozoon joins an ovum to form a zygote. (A zygote is a single cell, with a complete set of chromosomes, ...
of various organisms for identification purposes as in ''
Drosophila melanogaster
''Drosophila melanogaster'' is a species of fly (the taxonomic order Diptera) in the family Drosophilidae. The species is often referred to as the fruit fly or lesser fruit fly, or less commonly the "vinegar fly" or "pomace fly". Starting with Ch ...
'', 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
A regulatory sequence is a segment of a nucleic acid molecule which is capable of increasing or decreasing the expression of specific genes within an organism. Regulation of gene expression is an essential feature of all living organisms and vir ...
; 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.
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).
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 vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called "test-tube experiments", these studies in biology an ...
'' (in a dish), or even ''
in vivo
Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and ...
'' (in the living organism).
Genetically combining several spectral variants of GFP is a useful trick for the analysis of brain circuitry (
Brainbow
Brainbow is a process by which individual neurons in the brain can be distinguished from neighboring neurons using fluorescent proteins. By randomly expressing different ratios of red, green, and blue derivatives of green fluorescent protein in in ...
).
Other interesting uses of fluorescent proteins in the literature include using FPs as sensors of
neuron
A neuron, neurone, or nerve cell is an electrically excitable cell that communicates with other cells via specialized connections called synapses. The neuron is the main component of nervous tissue in all animals except sponges and placozoa. N ...
membrane potential
Membrane potential (also transmembrane potential or membrane voltage) is the difference in electric potential between the interior and the exterior of a biological cell. That is, there is a difference in the energy required for electric charges ...
,
tracking of
AMPA
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, better known as AMPA, is a compound that is a specific agonist for the AMPA receptor, where it mimics the effects of the neurotransmitter glutamate.
There are several types of glutamatergic ...
receptors on cell membranes,
viral entry
Viral entry is the earliest stage of infection in the viral life cycle, as the virus comes into contact with the host cell (biology), cell and introduces viral material into the cell. The major steps involved in viral entry are shown below. Desp ...
and the infection of individual
influenza
Influenza, commonly known as "the flu", is an infectious disease caused by influenza viruses. Symptoms range from mild to severe and often include fever, runny nose, sore throat, muscle pain, headache, coughing, and fatigue. These symptoms ...
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
Cryobiology is the branch of biology that studies the effects of low temperatures on living things within Earth's cryosphere or in science. The word cryobiology is derived from the Greek words κρῧος ryos "cold", βίος ios "life", and λό ...
as a
viability assay
A viability assay is an assay that is created to determine the ability of organs, cells or tissues to maintain or recover a state of survival. Viability can be distinguished from the all-or-nothing states of life and death by the use of a quanti ...
. Correlation of viability as measured by
trypan blue
Trypan blue is an azo dye. It is a direct dye for cotton textiles. In biosciences, it is used as a vital stain to selectively colour dead tissues or cells blue.
Live cells or tissues with intact cell membranes are not coloured. Since cells a ...
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.
Split GFP
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.
Macro-photography
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.
Transgenic pets
Alba
''Alba'' ( , ) is the Scottish Gaelic name for Scotland. It is also, in English language historiography, used to refer to the polity of Picts and Scottish people, Scots united in the ninth century as the Kingdom of Alba, until it developed i ...
, a green-fluorescent rabbit, was created by a French laboratory commissioned by
Eduardo Kac
Eduardo Kac dwardoʊ kæts; ĕd·wâr′·dō kăts(1962) is a contemporary artist of dual nationality (American and Brazilian) whose artworks span a wide range of practices, including performance art, poetry, holography, interactive art, digital ...
using GFP for purposes of art and social commentary. The US company Yorktown Technologies markets to aquarium shops green fluorescent
zebrafish
The zebrafish (''Danio rerio'') is a freshwater fish belonging to the minnow family ( Cyprinidae) of the order Cypriniformes. Native to South Asia, it is a popular aquarium fish, frequently sold under the trade name zebra danio (and thus often ...
(
GloFish
The GloFish is a patented and trademarked brand of genetically engineered fluorescent fish. They have been created from several different species of fish: zebrafish (''Danio rerio'') were the first GloFish available in pet stores, and recently te ...
) 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
National Taiwan University (NTU; ) is a public research university in Taipei, Taiwan.
The university was founded in 1928 during Japanese rule as the seventh of the Imperial Universities. It was named Taihoku Imperial University and served d ...
. A Japanese-American Team created green-fluorescent
cat
The cat (''Felis catus'') is a domestic species of small carnivorous mammal. It is the only domesticated species in the family Felidae and is commonly referred to as the domestic cat or house cat to distinguish it from the wild members of ...
s as proof of concept to use them potentially as model organisms for diseases, particularly
HIV
The human immunodeficiency viruses (HIV) are two species of ''Lentivirus'' (a subgroup of retrovirus) that infect humans. Over time, they cause acquired immunodeficiency syndrome (AIDS), a condition in which progressive failure of the immune ...
.
In 2009 a South Korean team from Seoul National University bred the first transgenic
beagles
The beagle is a breed of small scent hound, similar in appearance to the much larger foxhound. The beagle was developed primarily for hunting hare, known as beagling. Possessing a great sense of smell and superior tracking instincts, the b ...
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.
Art
Julian Voss-Andreae
Julian Voss-Andreae (born 15 August 1970) is a German sculptor living and working in the U.S.
Life
Voss-Andreae's full first name is Johann Julian, in honor of his ancestor, German pastor Johann Valentin Andreae.
According to an interview with th ...
, 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
The University of Washington (UW, simply Washington, or informally U-Dub) is a public research university in Seattle, Washington.
Founded in 1861, Washington is one of the oldest universities on the West Coast; it was established in Seattle a ...
's
Friday Harbor Laboratories
Friday Harbor Laboratories (FHL), is a marine biology field station of the University of Washington, located in Friday Harbor, San Juan Island, Washington, United States. Friday Harbor Labs is known for its intensive summer classes offered to co ...
.
See also
*
Protein tag
Protein tags are peptide sequences genetically grafted onto a recombinant protein. Tags are attached to proteins for various purposes. They can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or a ...
*
pGLO
The pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating genetically modified organisms. The plasmid contains several reporter genes, most notably the green fluorescent protein (GFP) and the ampicillin resistan ...
*
Yellow fluorescent protein
Yellow fluorescent protein (YFP) is a genetic mutant of green fluorescent protein (GFP) originally derived from the jellyfish ''Aequorea victoria''. Its excitation peak is 513 nm and its emission peak is 527 nm. Like the parent GFP, YFP ...
*
Genetically encoded voltage indicator
Genetically encoded voltage indicator (or GEVI) is a protein that can sense membrane potential in a cell and relate the change in voltage to a form of output, often fluorescent level. It is a promising optogenetic recording tool that enables exp ...
References
Further reading
* Popular science book describing history and discovery of GFP
*
External links
A comprehensive article on fluorescent proteins at Scholarpedia* Interactive Java applet demonstrating the chemistry behind th
Video of 2008 Nobel Prize lecture of Roger Tsien on fluorescent proteinsExcitation and emission spectra for various fluorescent proteinsGreen Fluorescent Protein Chem Soc Rev themed issuededicated to the 2008 Nobel Prize winners in Chemistry, Professors
Osamu Shimomura
was a Japanese organic chemist and marine biologist, and Professor Emeritus at Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts and Boston University School of Medicine. He was awarded the Nobel Prize in Chemistry in 2008 for the ...
,
Martin Chalfie
Martin Lee Chalfie (born January 15, 1947) is an American scientist. He is University Professor at Columbia University. He shared the 2008 Nobel Prize in Chemistry along with Osamu Shimomura and Roger Y. Tsien "for the discovery and development ...
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.
Green Fluorescent Proteinon FPbase, a fluorescent protein database
*
{{DEFAULTSORT:Green Fluorescent Protein
Protein methods
Recombinant proteins
Cell imaging
Protein imaging
*
Bioluminescence
Cnidarian proteins