Editing Green fluorescent protein (section)

== Background ==

[[Image:Aequorea victoria.jpg|thumb|200px|''Aequorea victoria'']]
[[File:Neural progenitors in the olfactory bulb.tif|thumb|200px|3D reconstruction of confocal image of VEGF-overexpressing neural progenitors (red) and GFP-positive control neural progenitor cells (green) in the rat olfactory bulb. RECA-1-positive blood vessels - blue color.]]

=== 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 the jellyfish ''Aequorea victoria'' and its properties studied by [[Osamu Shimomura]].<ref name=Shimomura_1962>{{cite journal | vauthors = Shimomura O, Johnson FH, Saiga Y | title = Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea | journal = [[Journal of Cellular and Comparative Physiology]] | volume = 59 | issue = 3 | pages = 223–39 | date = Jun 1962 | pmid = 13911999 | doi = 10.1002/jcp.1030590302 }}</ref>  In ''A. victoria'', GFP fluorescence occurs when [[aequorin]] interacts with [[calcium|Ca<sup>2+</sup>]] ions, inducing a blue glow. Some of this luminescent energy is transferred to the GFP, shifting the overall color towards green.<ref name=Morise_1974>{{cite journal | vauthors = Morise H, Shimomura O, Johnson FH, Winant J | title = Intermolecular energy transfer in the bioluminescent system of Aequorea | journal = [[Biochemistry (journal)|Biochemistry]] | volume = 13 | issue = 12 | pages = 2656–62 | date = Jun 1974 | pmid = 4151620 | doi = 10.1021/bi00709a028 }}</ref> 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''.<ref name=Prasher_1992>{{cite journal | vauthors = Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ | title = Primary structure of the Aequorea victoria green-fluorescent protein | journal = [[Gene (journal)|Gene]] | volume = 111 | issue = 2 | pages = 229–33 | date = Feb 1992 | pmid = 1347277 | doi = 10.1016/0378-1119(92)90691-H }}</ref>  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 ''[[Escherichia coli|E. coli]]'' and ''[[Caenorhabditis elegans|C. elegans]]'', publishing the results in ''[[Science (journal)|Science]]'' in 1994.<ref name=Chalfie_1994>{{cite journal | vauthors = Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC | s2cid = 9043327 | title = Green fluorescent protein as a marker for gene expression | journal = [[Science (journal)|Science]] | volume = 263 | issue = 5148 | pages = 802–5 | date = Feb 1994 | pmid = 8303295 | doi = 10.1126/science.8303295 | bibcode = 1994Sci...263..802C }}</ref> Frederick Tsuji's lab independently reported the expression of the recombinant protein one month later.<ref name=Inouye_1994>{{cite journal | vauthors = Inouye S, Tsuji FI | title = Aequorea green fluorescent protein. Expression of the gene and fluorescence characteristics of the recombinant protein | journal = [[FEBS Letters]] | volume = 341 | issue = 2–3 | pages = 277–80 | date = Mar 1994 | pmid = 8137953 | doi = 10.1016/0014-5793(94)80472-9 | doi-access = free | bibcode = 1994FEBSL.341..277I }}</ref>  Remarkably, the GFP molecule folded and was fluorescent at room temperature, without the need for exogenous cofactors specific to the jellyfish. Although this near-wtGFP was fluorescent, it had several drawbacks, including dual peaked excitation spectra, pH sensitivity, chloride sensitivity, poor fluorescence quantum yield, poor photostability and poor folding at {{convert|37|C}}.

The first reported crystal structure of a GFP was that of the S65T mutant by the Remington group in ''[[Science (journal)|Science]]'' in 1996.<ref name="Ormo_1996"/>  One month later, the Phillips group independently reported the wild-type GFP structure in ''Nature Biotechnology''.<ref name="Yang_1996"/> These crystal structures provided vital background on [[chromophore]] formation and neighboring residue interactions. Researchers have modified these residues by directed and random mutagenesis to produce the wide variety of GFP derivatives in use today. Further research into GFP has shown that it is resistant to detergents, proteases, [[guanidinium chloride]] (GdmCl) treatments, and drastic temperature changes.<ref name="Brejc_1997">{{cite journal | vauthors = Brejc K, Sixma TK, Kitts PA, Kain SR, Tsien RY, Ormö M, Remington SJ | title = Structural basis for dual excitation and photoisomerization of the ''Aequorea victoria'' green fluorescent protein | journal = [[Proceedings of the National Academy of Sciences of the United States of America]] | volume = 94 | issue = 6 | pages = 2306–2311 | date = March 1997 | pmid = 9122190 | pmc = 20083 | doi = 10.1073/pnas.94.6.2306 | bibcode = 1997PNAS...94.2306B | doi-access = free }}</ref>

=== GFP derivatives ===
[[Image:FPbeachTsien.jpg|thumb|200px|The diversity of genetic mutations is illustrated by this San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins (derived from GFP and dsRed).]]
Due to the potential for widespread usage and the evolving needs of researchers, many different mutants of GFP have been engineered.<ref name=Shaner_2005>{{cite journal | vauthors = Shaner NC, Steinbach PA, Tsien RY | title = A guide to choosing fluorescent proteins | journal = Nature Methods | volume = 2 | issue = 12 | pages = 905–9 | date = Dec 2005 | pmid = 16299475 | doi = 10.1038/nmeth819 | s2cid = 10024284 | url = http://tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Methods%20-%20Choosing%20fluorescent%20proteins.pdf }}</ref><ref>{{cite book | vauthors = Wilhelmsson M, Tor Y | title = Fluorescent Analogs of Biomolecular Building Blocks: Design and Applications | publisher = Wiley | location = New Jersey | year = 2016 | isbn = 978-1-118-17586-6 }}</ref> The first major improvement was a single point mutation (S65T) reported in 1995 in ''Nature'' by [[Roger Y. Tsien|Roger Tsien]].<ref name=Heim_1995>{{cite journal | vauthors = Heim R, Cubitt AB, Tsien RY | title = Improved green fluorescence | journal = Nature | volume = 373 | issue = 6516 | pages = 663–4 | date = Feb 1995 | pmid = 7854443 | doi = 10.1038/373663b0 | url = http://tsienlab.ucsd.edu/Publications/Heim%201995%20Nature%20-%20Improved%20GFP.PDF | bibcode = 1995Natur.373..663H | s2cid = 40179694 }}</ref> This mutation dramatically improved the spectral characteristics of GFP, resulting in increased fluorescence, photostability, and a shift of the major excitation peak to 488&nbsp;nm, with the peak emission kept at 509&nbsp;nm. This matched the spectral characteristics of commonly available [[Fluorescein|FITC]] filter sets, increasing the practicality of use by the general researcher. A 37&nbsp;°C folding efficiency (F64L) point mutant to this scaffold, yielding enhanced GFP (EGFP), was discovered in 1995 by the laboratories of Thastrup<ref name=Thastrup_1995>{{ cite patent | country = US | number = 6172188 | status = patent | title = Fluorescent Proteins | pubdate = 2001-01-09 | fdate = 1997-03-17 | pridate = 1995-09-22 | inventor = Thastrup O, Tullin S, Kongsbak Poulsen L, Bjørn S }}</ref> and Falkow.<ref name=Cormack_1996>{{cite journal | vauthors = Cormack BP, Valdivia RH, Falkow S | title = FACS-optimized mutants of the green fluorescent protein (GFP) | journal = Gene | volume = 173 | issue = 1 Spec No | pages = 33–38 | date = 1996 | pmid = 8707053 | doi = 10.1016/0378-1119(95)00685-0 | doi-access = free }}</ref> EGFP allowed the practical use of GFPs in mammalian cells. EGFP has an [[Molar attenuation coefficient|extinction coefficient]] (denoted ε) of 55,000 M<sup>−1</sup>cm<sup>−1</sup>.<ref>{{cite journal | vauthors = McRae SR, Brown CL, Bushell GR | title = Rapid purification of EGFP, EYFP, and ECFP with high yield and purity | journal = Protein Expression and Purification | volume = 41 | issue = 1 | pages = 121–127 | date = May 2005 | pmid = 15802229 | doi = 10.1016/j.pep.2004.12.030 }}</ref> The fluorescence [[quantum yield]] (QY) of EGFP is 0.60. The relative brightness, expressed as ε•QY, is 33,000 M<sup>−1</sup>cm<sup>−1</sup>.

Superfolder GFP (sfGFP), a series of mutations that allow GFP to rapidly fold and mature even when fused to poorly folding peptides, was reported in 2006.<ref name="Pedelacq_2006">{{cite journal | vauthors = Pédelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS | title = Engineering and characterization of a superfolder green fluorescent protein | journal = Nature Biotechnology | volume = 24 | issue = 1 | pages = 79–88 | date = Jan 2006 | pmid = 16369541 | doi = 10.1038/nbt1172 | s2cid = 2966399 }}</ref>

Many other mutations have been made, including color mutants; in particular, blue fluorescent protein (EBFP, EBFP2, Azurite, mKalama1), cyan fluorescent protein (ECFP, Cerulean, CyPet, 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&nbsp;nm. Therefore, they can be used as Zn biosensor.<ref name="Barondeau_2002">{{cite journal|author-link4=Elizabeth D. Getzoff | vauthors = Barondeau DP, Kassmann CJ, Tainer JA, Getzoff ED | title = Structural chemistry of a green fluorescent protein Zn biosensor | journal = Journal of the American Chemical Society | volume = 124 | issue = 14 | pages = 3522–3524 | date = Apr 2002 | pmid = 11929238 | doi = 10.1021/ja0176954 | bibcode = 2002JAChS.124.3522B }}</ref>

[[Image:174-GFPLikeProteins GFP-like Proteins.tif|thumb|left|A palette of variants of GFP and DsRed.]]
Chromophore binding. The critical mutation in cyan derivatives is the Y66W substitution, which causes the chromophore to form with an [[indole]] rather than phenol component. Several additional compensatory mutations in the surrounding barrel are required to restore brightness to this modified chromophore due to the increased bulk of the indole group. In ECFP and Cerulean, the N-terminal half of the seventh strand exhibits two conformations. These conformations both have a complex set of van der Waals interactions with the chromophore. The Y145A and H148D mutations in Cerulean stabilize these interactions and allow the chromophore to be more planar, better packed, and less prone to collisional quenching.<ref name=Lelimousin_2009>{{cite journal | vauthors = Lelimousin M, Noirclerc-Savoye M, Lazareno-Saez C, Paetzold B, Le Vot S, Chazal R, Macheboeuf P, Field MJ, Bourgeois D, Royant A | title = Intrinsic dynamics in ECFP and Cerulean control fluorescence quantum yield | journal = Biochemistry | volume = 48 | issue = 42 | pages = 10038–10046 | date = Oct 2009 | pmid = 19754158 | doi = 10.1021/bi901093w }}</ref>

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.<ref>{{cite journal | vauthors = Goedhart J, von Stetten D, Noirclerc-Savoye M, Lelimousin M, Joosen L, Hink MA, van Weeren L, Gadella TW, Royant A | title = Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93% | journal = Nature Communications | volume = 3 | pages = 751 | date = 2012 | pmid = 22434194 | pmc = 3316892 | doi = 10.1038/ncomms1738 | bibcode = 2012NatCo...3..751G }}</ref> The red-shifted wavelength of the YFP derivatives is accomplished by the T203Y mutation and is due to π-electron stacking interactions between the substituted tyrosine residue and the chromophore.<ref name="Tsien_1998" /> These two classes of spectral variants are often employed for [[Förster resonance energy transfer#CFP-YFP pairs|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.<ref name=Miesenbock_1998>{{cite journal | vauthors = Miesenböck G, De Angelis DA, Rothman JE | title = Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins | journal = Nature | volume = 394 | issue = 6689 | pages = 192–5 | date = Jul 1998 | pmid = 9671304 | doi = 10.1038/28190 | bibcode = 1998Natur.394..192M | s2cid = 4320849 }}</ref>

Redox sensitive GFP ([[roGFP]]) was engineered by introduction of cysteines into the beta barrel structure. The [[redox]] state of the cysteines determines the [[fluorescent]] properties of [[roGFP]].<ref name=Hanson_2004>{{cite journal | vauthors = Hanson GT, Aggeler R, Oglesbee D, Cannon M, Capaldi RA, Tsien RY, Remington SJ | title = Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators | journal = The Journal of Biological Chemistry | volume = 279 | issue = 13 | pages = 13044–53 | date = Mar 2004 | pmid = 14722062 | doi = 10.1074/jbc.M312846200 | doi-access = free }}</ref>

=== 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]] that causes the GFP to bind to [[cell membrane]]s. However, the same term is also used to refer to [[monomer]]ic GFP, which is often achieved by the dimer interface breaking A206K mutation.<ref name="Zacharias_2002">{{cite journal | vauthors = Zacharias DA, Violin JD, Newton AC, Tsien RY | s2cid = 14957077 | title = Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells | journal = Science | volume = 296 | issue = 5569 | pages = 913–16 | date = May 2002 | pmid = 11988576 | doi = 10.1126/science.1068539 | bibcode = 2002Sci...296..913Z }}</ref>  Wild-type GFP has a weak [[protein dimer|dimer]]ization tendency at concentrations above 5&nbsp;mg/mL. mGFP also stands for "modified GFP," which has been optimized through amino acid exchange for stable expression in plant cells.