CH391L/S12/Fluorescent Proteins: Difference between revisions
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GFP was first discovered by [[Osamu Shimomura]] in ''Aequorea'' jellyfish as a companion protein to the aequorin responsible for the blue glow of the organism.<cite>Shimomura1962</cite><cite>Tsien1998</cite> Shimomura and his group further characterized and identified the peak luminescence of GFP as similar to that of ''Aequorea'' tissue, both of which differed from the peak of the aequorin protein significantly, indicating that GFP altered the color of the aequorin from its natural blue to the green expressed by the organism. They showed that the mechanism for this was transfer of energy from the aequorin to GFP in the presence of a cation<cite>Morise1974</cite> The crucial breakthrough came when [[Douglas Prasher]] et al cloned the gene and identified its amino acid and DNA sequence.<cite>Prasher1992</cite> Further characterization showed that expression of the gene led to luminescence in other organism, providing the key inference that '''all of the information necessary for post-translational synthesis of the chromophore was in the gene itself, and no jellyfish-specific enzymes were needed for production of functional GFP.''' | GFP was first discovered by [[Osamu Shimomura]] in ''Aequorea'' jellyfish as a companion protein to the aequorin responsible for the blue glow of the organism.<cite>Shimomura1962</cite><cite>Tsien1998</cite> Shimomura and his group further characterized and identified the peak luminescence of GFP as similar to that of ''Aequorea'' tissue, both of which differed from the peak of the aequorin protein significantly, indicating that GFP altered the color of the aequorin from its natural blue to the green expressed by the organism. They showed that the mechanism for this was transfer of energy from the aequorin to GFP in the presence of a cation<cite>Morise1974</cite> The crucial breakthrough came when [[Douglas Prasher]] et al cloned the gene and identified its amino acid and DNA sequence.<cite>Prasher1992</cite> Further characterization showed that expression of the gene led to luminescence in other organism, providing the key inference that '''all of the information necessary for post-translational synthesis of the chromophore was in the gene itself, and no jellyfish-specific enzymes were needed for production of functional GFP.''' | ||
=== Structure === | === Structure and Characterization === | ||
GFP consists of a single β-sheet with alpha helices containing the covalently bonded [[chromophore]] 4-(p-hydroxybenzylidene)imidazolidin-5-one (HBI) running through the center. Wild type GFP has been shown to have 2 excitation peaks at 395-397nm and 470-475nm. The emission spectrum of wild type GFP has a single peak at 504nm. | |||
GFP consists of a single β-sheet with alpha helices containing the covalently bonded [[chromophore]] 4-(p-hydroxybenzylidene)imidazolidin-5-one (HBI) running through the center. | |||
[[Image:GFP and fluorophore.png|thumb|200px|GFP molecules drawn in cartoon style, one fully and one with the side of the [[beta barrel]] cut away to reveal the [[chromophore]]]] | [[Image:GFP and fluorophore.png|thumb|200px|GFP molecules drawn in cartoon style, one fully and one with the side of the [[beta barrel]] cut away to reveal the [[chromophore]]]] | ||
=== GFP Derivatives (excluding EGFP and destabilized GFPS) === | |||
As of 1998, there were 7 main classes of GFP: | |||
*wild-type mixture of neutral phenol and anionic phenolate (above) | |||
*phenolate anion | |||
*neutral phenol | |||
*phenolate anion with stacked pi electron system | |||
*indole | |||
*imidazole | |||
*phenyl. | |||
The excitation and emission wavelength spectra of each of these derivatives are different, as shown in the table. | |||
The first 4 classes are polypeptides with a Tyr and position 66, while the final 3 have Trp, His and Phe at that position respectively.<cite>Tsien1998</cite> | |||
[[Image:GFPemissiontable.jpg|thumb|200px|Table of various GFP mutants' emission color, excitation wavelength peaks, and emission wavelength peaks.]] | |||
==References== | ==References== | ||
Revision as of 21:52, 18 March 2012
Green Fluorescent Protein (GFP)
History
GFP was first discovered by Osamu Shimomura in Aequorea jellyfish as a companion protein to the aequorin responsible for the blue glow of the organism.[1][2] Shimomura and his group further characterized and identified the peak luminescence of GFP as similar to that of Aequorea tissue, both of which differed from the peak of the aequorin protein significantly, indicating that GFP altered the color of the aequorin from its natural blue to the green expressed by the organism. They showed that the mechanism for this was transfer of energy from the aequorin to GFP in the presence of a cation[3] The crucial breakthrough came when Douglas Prasher et al cloned the gene and identified its amino acid and DNA sequence.[4] Further characterization showed that expression of the gene led to luminescence in other organism, providing the key inference that all of the information necessary for post-translational synthesis of the chromophore was in the gene itself, and no jellyfish-specific enzymes were needed for production of functional GFP.
Structure and Characterization
GFP consists of a single β-sheet with alpha helices containing the covalently bonded chromophore 4-(p-hydroxybenzylidene)imidazolidin-5-one (HBI) running through the center. Wild type GFP has been shown to have 2 excitation peaks at 395-397nm and 470-475nm. The emission spectrum of wild type GFP has a single peak at 504nm.
GFP Derivatives (excluding EGFP and destabilized GFPS)
As of 1998, there were 7 main classes of GFP:
- wild-type mixture of neutral phenol and anionic phenolate (above)
- phenolate anion
- neutral phenol
- phenolate anion with stacked pi electron system
- indole
- imidazole
- phenyl.
The excitation and emission wavelength spectra of each of these derivatives are different, as shown in the table. The first 4 classes are polypeptides with a Tyr and position 66, while the final 3 have Trp, His and Phe at that position respectively.[2]
References
- SHIMOMURA O, JOHNSON FH, and SAIGA Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol. 1962 Jun;59:223-39. DOI:10.1002/jcp.1030590302 |
- Tsien RY. The green fluorescent protein. Annu Rev Biochem. 1998;67:509-44. DOI:10.1146/annurev.biochem.67.1.509 |
- Morise H, Shimomura O, Johnson FH, and Winant J. Intermolecular energy transfer in the bioluminescent system of Aequorea. Biochemistry. 1974 Jun 4;13(12):2656-62. DOI:10.1021/bi00709a028 |
- Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, and Cormier MJ. Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992 Feb 15;111(2):229-33. DOI:10.1016/0378-1119(92)90691-h |
