User:Laura Carbone/Sandbox 1: Difference between revisions
From Proteopedia
Jump to navigationJump to search
No edit summary |
No edit summary |
||
| Line 1: | Line 1: | ||
[[Image:Green1ema.png|center|300px]] | [[Image:Green1ema.png|center|300px]] | ||
'''Green fluorescent protein (GFP)''' is a [http://en.wikipedia.org/wiki/Bioluminescent bioluminescent] polypeptide consisting of 238 residues isolated from the body of [http://en.wikipedia.org/wiki/Aequorea_victoria ''Aequorea victoria''] jellyfish.<ref name="PDBsum">[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1ema&template=main.html], Protein Database (PDBsum): 1ema. European Bioinformatics (EBI); 2009.</ref> GFP converts the blue chemiluminescent of [http://en.wikipedia.org/wiki/Aequorin aequorin] in the jellyfish into green fluorescent light.<ref name="Yang">[http://www-bioc.rice.edu/Bioch/Phillips/Papers/gfpbio.html], Yang F, Moss LG, Phillips GN Jr. 1996. The molecular structure of green fluorescent protein. Biotechnology. 14: 1246-1251. DOI 10.1038/nbt1096-1246.</ref> In the laboratory, GFP can be incorporated into a variety of biological systems in order to function as a marker protein. Since its discovery in 1962, GFP has become a significant contributor to the research of monitoring gene expression, localization, mobility, traffic, interactions between various membrane and cytoplasmic proteins, as well as many others.<ref name="Haldar">[http://www.springerlink.com/content/wvg513864266g77n/fulltext.pdf], Haldar S, Chattopadhyay A. 2009. The green journey. J Fluoresc. 19:1-2. DOI 10.1007/s10895-008-0455-6.</ref> | '''Green fluorescent protein (GFP)''' is a [http://en.wikipedia.org/wiki/Bioluminescent bioluminescent] polypeptide consisting of 238 residues isolated from the body of [http://en.wikipedia.org/wiki/Aequorea_victoria ''Aequorea victoria''] jellyfish.<ref name="PDBsum">[http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=1ema&template=main.html], Protein Database (PDBsum): 1ema. European Bioinformatics (EBI); 2009.</ref> GFP converts the blue chemiluminescent of [http://en.wikipedia.org/wiki/Aequorin aequorin] in the jellyfish into green fluorescent light.<ref name="Yang">[http://www-bioc.rice.edu/Bioch/Phillips/Papers/gfpbio.html], Yang F, Moss LG, Phillips GN Jr. 1996. The molecular structure of green fluorescent protein. Biotechnology. 14: 1246-1251. DOI 10.1038/nbt1096-1246.</ref> In remains unclear why these jellyfish use fluorescence, why green is better than blue, or why they produce a separate protein for green fluorescence as opposed to simply mutating the present aequorin to shift its wavelength,<ref name="Tsien" /> but in the laboratory, GFP can be incorporated into a variety of biological systems in order to function as a marker protein. Since its discovery in 1962, GFP has become a significant contributor to the research of monitoring gene expression, localization, mobility, traffic, interactions between various membrane and cytoplasmic proteins, as well as many others.<ref name="Haldar">[http://www.springerlink.com/content/wvg513864266g77n/fulltext.pdf], Haldar S, Chattopadhyay A. 2009. The green journey. J Fluoresc. 19:1-2. DOI 10.1007/s10895-008-0455-6.</ref> | ||
==History== | ==History== | ||
| Line 8: | Line 8: | ||
Shimomura was originally looking only to isolate the blue luminescent protein of ''Aequorea victoria'', traditionally thought to be [http://en.wikipedia.org/wiki/Luciferase luciferase], but it would soon become apparent that the glow was in fact due to aequorin, a substance related, but slightly varying from luciferase.<ref name="Haldar" /><ref name="Shimomura" /> However, the light emitted from aequorin still differed from the light emitted from the wild jellyfish. This quandary led to the discovery of the green fluorescent protein responsible for this disparity, but sufficient amounts of the protein could not be collected for study until 1979. The journey to discover the nature of GFP had begun.<ref name="Shimomura" /> | Shimomura was originally looking only to isolate the blue luminescent protein of ''Aequorea victoria'', traditionally thought to be [http://en.wikipedia.org/wiki/Luciferase luciferase], but it would soon become apparent that the glow was in fact due to aequorin, a substance related, but slightly varying from luciferase.<ref name="Haldar" /><ref name="Shimomura" /> However, the light emitted from aequorin still differed from the light emitted from the wild jellyfish. This quandary led to the discovery of the green fluorescent protein responsible for this disparity, but sufficient amounts of the protein could not be collected for study until 1979. The journey to discover the nature of GFP had begun.<ref name="Shimomura" /> | ||
In the 1990’s, [http://en.wikipedia.org/wiki/Douglas_Prasher Douglas Prasher], Frank Predergast, and co-workers successfully cloned the gene that encoded for GFP. [http://en.wikipedia.org/wiki/Martin_Chalfie Martin Chalfie] further pursued this line of work and was eventually able to express GFP in heterologous systems such as E. coli and C. elegans. Chalfie’s research provided the first evidence that GFP was unique as it did not require the presence of any exogenous substance or cofactor for fluorescence.<ref name="Haldar" /> | In the 1990’s, [http://en.wikipedia.org/wiki/Douglas_Prasher Douglas Prasher], Frank Predergast, and co-workers successfully cloned the gene that encoded for GFP. [http://en.wikipedia.org/wiki/Martin_Chalfie Martin Chalfie] further pursued this line of work and was eventually able to express GFP in heterologous systems such as E. coli and C. elegans. Chalfie’s research provided the first evidence that GFP was unique as it did not require the presence of any exogenous substance or cofactor for fluorescence.<ref name="Haldar" /> The lack for the need for a cofactor proved that the cloned GFP gene contained all the information necessary for posttranslational synthesis of the chromophore. <ref name="Tsien" /> | ||
[http://en.wikipedia.org/wiki/Roger_Tsien Roger Tsien] and co-workers were intrigued by the absence of a necessary cofactor and began to research the structure of GFP and how it relates to its fluorescence. They discovered that a helix within the beta barrel structure of GFP actually contained a fluorophore responsible for fluorescence. In researching its structure, they were able to develop GFP derivatives with improved fluorescence and photo-stability. Shimomura, Chalfie, and Tsien were each recognized for their work involving GFP with the Nobel Prize in 2008.<ref name="Haldar" /> In the time since the work of these three researchers, GFP has been successfully expressed and utilized in bacteria, yeast, slime mold, plants, drosophila fruit flies, zebra-fish, and mammalian cells.<ref name="Yang" /> Below, mice have had GFP inserted into their genomes for studies in neurology. | [http://en.wikipedia.org/wiki/Roger_Tsien Roger Tsien] and co-workers were intrigued by the absence of a necessary cofactor and began to research the structure of GFP and how it relates to its fluorescence. They discovered that a helix within the beta barrel structure of GFP actually contained a fluorophore responsible for fluorescence. In researching its structure, they were able to develop GFP derivatives with improved fluorescence and photo-stability. Shimomura, Chalfie, and Tsien were each recognized for their work involving GFP with the Nobel Prize in 2008.<ref name="Haldar" /> In the time since the work of these three researchers, GFP has been successfully expressed and utilized in bacteria, yeast, slime mold, plants, drosophila fruit flies, zebra-fish, and mammalian cells.<ref name="Yang" /> Below, mice have had GFP inserted into their genomes for studies in neurology. | ||
| Line 38: | Line 38: | ||
===The Chromophore=== | ===The Chromophore=== | ||
The <scene name='User:Laura_Carbone/Sandbox_1/Chromophore/5' target='2'>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<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" /> | The <scene name='User:Laura_Carbone/Sandbox_1/Chromophore/5' target='2'>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 at 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='User:Laura_Carbone/Sandbox_1/Chromophore_structure/3' target='2'>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> | 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='User:Laura_Carbone/Sandbox_1/Chromophore_structure/3' target='2'>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> | ||
