Editing Green fluorescent protein (section)

== Structure ==
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.<ref name="Tsien_1998"/><ref name=Ormo_1996>{{cite journal | vauthors = Ormö M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ | s2cid = 43030290 | title = Crystal structure of the Aequorea victoria green fluorescent protein | journal = Science | volume = 273 | issue = 5280 | pages = 1392–5 | date = Sep 1996 | pmid = 8703075 | doi = 10.1126/science.273.5280.1392 | bibcode = 1996Sci...273.1392O }}</ref><ref name=Yang_1996>{{cite journal | vauthors = Yang F, Moss LG, Phillips GN | title = The molecular structure of green fluorescent protein | journal = Nature Biotechnology | volume = 14 | issue = 10 | pages = 1246–51 | date = Oct 1996 | pmid = 9631087 | doi = 10.1038/nbt1096-1246 | url = https://scholarship.rice.edu/bitstream/1911/19233/1/9727628.PDF | hdl = 1911/19233 | s2cid = 34713931 | hdl-access = free }}</ref> 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<ref name="Brejc_1997" />),<ref name="Ormo_1996"/> creating what is referred to as a "β-can" formation, which is unique to the GFP-like family.<ref name="Yang_1996"/> 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.<ref name=Bokman_1981>{{cite journal | vauthors = Bokman SH, Ward WW | title = Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein | journal = Biochemistry | volume = 21 | issue = 19 | pages = 4535–4540 | date = 1982 | doi = 10.1021/bi00262a003| pmid = 6128025 }}</ref> 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''.<ref name="pmid18759496">{{cite journal | vauthors = Pouwels LJ, Zhang L, Chan NH, Dorrestein PC, Wachter RM | title = Kinetic isotope effect studies on the de novo rate of chromophore formation in fast- and slow-maturing GFP variants | journal = Biochemistry | volume = 47 | issue = 38 | pages = 10111–22 | date = Sep 2008 | pmid = 18759496 | pmc = 2643082 | doi = 10.1021/bi8007164 }}</ref>  The hydrogen-bonding network and electron-stacking interactions with these sidechains influence the color, intensity and photostability of GFP and its numerous derivatives.<ref>{{cite journal | vauthors = Chudakov DM, Matz MV, Lukyanov S, Lukyanov KA | s2cid = 10767597 | title = Fluorescent proteins and their applications in imaging living cells and tissues | journal = Physiological Reviews | volume = 90 | issue = 3 | pages = 1103–63 | date = Jul 2010 | pmid = 20664080 | doi = 10.1152/physrev.00038.2009 }}</ref> 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.<ref name="Brejc_1997" /> 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.<ref name="pmid18470931">{{cite journal | vauthors = Stepanenko OV, Verkhusha VV, Shavlovsky MM, Kuznetsova IM, Uversky VN, Turoverov KK | title = Understanding the role of Arg96 in structure and stability of green fluorescent protein | journal = Proteins | volume = 73 | issue = 3 | pages = 539–551 | date = November 2008 | pmid = 18470931 | pmc = 2908022 | doi = 10.1002/prot.22089 }}</ref>
[[File:GFP Fluorescent Protein Movie.gif|thumb|377x377px|GFP Movie showing entire structure and zoom in to fluorescent chromophore.]]
{|
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|[[Image:Gfp and fluorophore.png|thumb|300px|GFP molecules drawn in cartoon style, one fully and one with the side of the [[beta barrel]] cut away to reveal the [[chromophore]] (highlighted as [[Ball-and-stick model|ball-and-stick]]). From {{PDB|1GFL}}.]]
|[[Image:GFP structure.png|thumb|200px|GFP [[ribbon diagram]]. From {{PDB|1EMA}}.]]
|}
[[File:Final Y66 and Y145 1EMA structure of GFP.png|thumb|Structure of GFP with PDB code 1EMA. The two tyrosine residues that undergo substitution mutations at positions 66 and 145 are highlighted in red. These two tyrosines at position 66 and 145 need to be substituted with histidine and phenylalanine, respectively for this protein to fluoresce blue.]]

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

=== Autocatalytic formation of the chromophore in wtGFP ===
{{hatnote|For a synthetic analogue see also [[3,5-Difluoro-4-hydroxybenzylidene imidazolinone]].}}
[[File:GFP mechanism.svg|1200x1200px]]

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.<ref name=":2">{{cite journal | vauthors = Rosenow MA, Huffman HA, Phail ME, Wachter RM | title = The crystal structure of the Y66L variant of green fluorescent protein supports a cyclization-oxidation-dehydration mechanism for chromophore maturation | journal = Biochemistry | volume = 43 | issue = 15 | pages = 4464–4472 | date = April 2004 | pmid = 15078092 | doi = 10.1021/bi0361315 }}</ref> The formed HBI fluorophore is highlighted in green.

The reactions are catalyzed by residues Glu222 and Arg96.<ref name=":2" /><ref>{{Cite journal| vauthors = Ma Y, Yu JG, Sun Q, Li Z, Smith SC |date=2015-07-01|title=The mechanism of dehydration in chromophore maturation of wild-type green fluorescent protein: A theoretical study |journal=Chemical Physics Letters|language=en|volume=631-632|pages=42–46|doi=10.1016/j.cplett.2015.04.061|bibcode=2015CPL...631...42M |issn=0009-2614}}</ref> An analogous mechanism is also possible with threonine in place of Ser65.