Green Fluorescent Protein: Difference between revisions
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Green fluorescent protein (<scene name='Green_Fluorescent_Protein/Initial/1'>default scene</scene>) is a 21 kDa protein consisting of 238 residues strung together to form a | Green fluorescent protein (<scene name='Green_Fluorescent_Protein/Initial/1'>default scene</scene>) is a 21 kDa protein consisting of 238 residues strung together to form a | ||
<scene name='Green_Fluorescent_Protein/Secondary_structure/1'>secondary structure</scene> of five α-helices and one eleven-stranded β-pleated sheet,<ref name="PDBsum" /> where each strand contains nine to thirteen residues each.<ref name="Ormo" /> (To view the primary and secondary structure of GFP, go to [[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1ema&template=protein.html&r=wiring&l=1&chain=A www.ebi.aci.uk]].) These β-strands display an almost “seamless symmetry” in which only two of the strands vary in structural content.<ref name="Phillips">Phillips GN Jr. 1997. Structure and dynamics of green fluorescent protein. Curr Opin Struct Biol. 7(6):821-827. DOI 10.1016/S0959-440X(97)80153-4.</ref> This β-sheet conforms itself through regular hydrogen bonding into a β-barrel.<ref name="Yang" /> In GFP, the structure is so regular that <scene name='Green_Fluorescent_Protein/Water_stripes/1'>"stripes"</scene> of water molecules (red) can be seen following the structure of the barrel.<ref name="Phillips" /> Together with the α-helices at either end of the molecule, a nearly perfect cylinder is produced, 42Å long and 24Å in diameter,<ref name="Ormo" /> creating what is referred to as a “β-can” formation.<ref name="Phillips" /> The short helical segments at either end of the cylinder form “caps” to further protect the interior of the β-barrel.<ref name="Phillips" /> Overall stability is maintained by this β-can structure, helping to resist unfolding from heat and other denaturants.<ref name="Yang" /> | <scene name='Green_Fluorescent_Protein/Secondary_structure/1'>secondary structure</scene> of five α-helices and one eleven-stranded β-pleated sheet,<ref name="PDBsum" /> where each strand contains nine to thirteen residues each.<ref name="Ormo" /> (To view the primary and secondary structure of GFP, go to [[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1ema&template=protein.html&r=wiring&l=1&chain=A www.ebi.aci.uk]].) These β-strands display an almost “seamless symmetry” in which only two of the strands vary in structural content.<ref name="Phillips">Phillips GN Jr. 1997. Structure and dynamics of green fluorescent protein. Curr Opin Struct Biol. 7(6):821-827. DOI 10.1016/S0959-440X(97)80153-4.</ref> This β-sheet conforms itself through regular hydrogen bonding into a β-barrel.<ref name="Yang" /> In GFP, the structure is so regular that <scene name='Green_Fluorescent_Protein/Water_stripes/1'>"stripes"</scene> of water molecules (red) can be seen following the structure of the barrel.<ref name="Phillips" /> Together with the α-helices at either end of the molecule, a nearly perfect cylinder is produced, 42Å long and 24Å in diameter,<ref name="Ormo" /> creating what is referred to as a “β-can” formation.<ref name="Phillips" /> The short helical segments at either end of the cylinder form “caps” to further protect the interior of the β-barrel.<ref name="Phillips" /> Overall stability is maintained by this β-can structure, helping to resist unfolding from heat and other denaturants.<ref name="Yang" /> | ||
One <scene name='Green_Fluorescent_Protein/Central_helix/1'>α-helix</scene> can be found running through the central axis of the β-barrel,<ref name="Haldar" /> roughly <scene name='Green_Fluorescent_Protein/Perpendicular/1'>perpendicular</scene> to the symmetry axis of the barrel.<ref name="Ormo">Ormo M, Cubitt AB, Kallio K, Gross LA, Tsien RY, Remington SJ. 1996. Crystal structure of the ''Aequorea victoria'' green fluorescent protein. Science. 273(5280):1392-1395. DOI 10.1126/science.273.5280.1392.</ref> This helix is extremely important as it contains the fluorophore responsible for fluorescence.<ref name="Yang" /><ref name="Haldar" /> This α-helix in particular is highly stabilized by the many <scene name='Green_Fluorescent_Protein/Spacefill/1'>contacts</scene> that are made with each strand of the barrel.<ref name="Andrews">Andrews BT, Gosavi S, Finke JM, Onuchic JN, Jennings PA. 2008. The dual-basin landscape in GFP. Proceedings of the National Academy of Sciences. 105(34):12283-12288. DOI 10.1073/pnas.0804039105.</ref> | |||
===The Chromophore=== | |||
The <scene name='Green_Fluorescent_Protein/Chromophore/1'>chromophore</scene> of GFP is located at the center of the β-barrel with a wild-type excitation peak of 395 nm, and a minor peak at 475 nm (about three times less intense<ref name="Tsien" />) <ref name="Yang" /><ref name="Cubitt" /><ref name="Ormo" /><ref name="Phillips" /> with extinction coefficients of approximately 30,000 and 7,000 M<sup>-1</sup> cm<sup>-1</sup>, respectively.<ref name="Yang" /><ref name="Phillips" /> Interestingly, the ''Aequorea victoria'' jellyfish utilizes the smaller of the two excitation peaks as pure aequorin emits a light of 470 nm.<ref name="Tsien">Tsien, Roger Y. 1998. The Green Fluorescent Protein. Annual Review in Biochemistry. 67:509-544.</ref> The relative amplitudes of these two excitation peaks can vary depending on environmental factors and previous illumination.<ref name="Ormo" /> For example, continued excitation leads to a diminution of the 395 nm excitation peak with a reciprocal amplification of the 475 nm peak.<ref name="Phillips" /> Regardless of absorption, the chromophore of GFP emits light of 508 nm.<ref name="Yang" /><ref name="Cubitt" /><ref name="Ormo" /><ref name="Phillips" /> | |||
Three amino residues in the central α-helix constitute the fluorophore of GFP: Ser<sup>65</sup>Tyr<sup>66</sup>Gly<sup>67</sup> (see left). Tsien et al. discovered that this tri-peptide sequence is post-translationally modified by internal cyclization and oxidation<ref name="Haldar" /> to produce a <scene name='Green_Fluorescent_Protein/Chromophore_structure/1'>4-(p-hydroxybenzylidene)-imidazolidin-5-one</scene> structure.<ref name="Yang" /> Studies with E. coli proposed a sequential mechanism for the formation of the fluorophore that was initiated by a rapid cyclization between Ser<sup>65</sup> and Gly<sup>67</sup> to form an imidazolin-5-one intermediate.<ref name="Yang" /> This rapid cyclization is carried out via nucleophilic attack of the amino group from Gly<sup>67</sup> on the carbonyl group of Ser<sup>65</sup> to form a five-membered ring. The loss of water then forms the imidazolin-5-one intermediate.<ref name="Cubitt" /> Cyclization is succeeded by a much slower rate-limiting oxygenation of the Tyr<sup>66</sup> hydroxybenzyl side chain by atmospheric oxygen (No fluorescence was seen in anaerobically grown E. coli.), resulting in the 4-(p-hydroxybenzylidene)-imidazolidin-5-one stucture.<ref name="Yang" /><ref name="Cubitt" /><ref name="Phillips" /> The double bond that results from this series of reactions results in the linkage of the two π-systems of the rings, forming a larger conjugated system essential for fluorophore stability. <ref name="Bublitz"> Bublitz G, King BA, Boxer SG. 1998. Electronic structure of the chromophore in green fluorescent protein (GFP). Journal of the American Chemical Society. 120(36): 9370-9371. DOI 10.1021/ja98160e.</ref> | |||
[[Image:GFP Chromophore.png|center|489x360px]] | |||
The process is completely auto-catalytic such that there are no known co-factors or enzymatic components required.<ref name="Yang" /> Despite the stability of the final product, while the chromophore is forming, the environmental temperature cannot drop below 30°C or the yield of viable GFP will decrease substantially.<ref name="Yang" /><ref name="Phillips" /> This, of course, is not an issue for the protein in nature as the jellyfish is unlikely to encounter waters of this degree in the Pacific Northwest.<ref name="Tsien" /> Such a temperature sensitivity is only relevant during formation as the stability of the final product is maintained through a network of close contacts surrounding the fluorophore.<ref name="Yang" /> This, however, can and has been used in in [http://en.wikipedia.org/wiki/Pulse-chase_analysis pulse-chase experiments] in which the GFP-expressing cells are exposed to varying temperatures in place of labeled vs. unlabeled trials.<ref name="Tsien" /> | |||
As the central α-helix is not located directly in the center of the β-barrel, cavities of differing area exist on either side of the chromophore. The larger cavity, consisting of about 135 Å,<ref name="Ormo" /> does not open out to the bulk solvent, but rather houses <scene name='Green_Fluorescent_Protein/Water_molecules/1' target='2'>four water molecules</scene>.<ref name="Ormo" /><ref name="Van" /> Had this space not been occupied, it would be expected to considerably destabilize the protein as a whole. The hydrogen bonding created by the presence of the water molecules, however, helps to link the buried <scene name='Green_Fluorescent_Protein/Gln69_glu222/1' target='2'>side chains</scene> of Glu<sup>222</sup> and Gln<sup>69</sup> that would otherwise be actively polar.<ref name="Ormo" /> Therefore, the water molecules are extremely important in establishing a hydrogen bonding network about the chromophor.<ref name="Lammich"> Lammich L, Petersen MA, Nielsen, MB, Andersen LH. 2007. The gas-phase absorption spectrum of a neutral GFP model chromophore. Biophysical Journal. 92: 201-207. DOI 10.1529/biophysj.106.093674.</ref> | |||
