Green Fluorescent Protein: Difference between revisions
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The <scene name='Green_Fluorescent_Protein/Chromophore/1'>chromophore</scene> (<scene name='Green_Fluorescent_Protein/1ema_gfp_chromophorezoom/6'>top view</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" /> | The <scene name='Green_Fluorescent_Protein/Chromophore/1'>chromophore</scene> (<scene name='Green_Fluorescent_Protein/1ema_gfp_chromophorezoom/6'>top view</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 | 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 below). 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]] | [[Image:GFP Chromophore.png|center|489x360px]] | ||
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This quandary led Phillips to study the acid/base chemistry catalyzing the initial cyclization of the chromophore. He found that Arg<sup>96</sup> actually acts as a | This quandary led Phillips to study the acid/base chemistry catalyzing the initial cyclization of the chromophore. He found that Arg<sup>96</sup> actually acts as a | ||
<scene name='Green_Fluorescent_Protein/Arg96/1' target='3' >base</scene> by withdrawing electrons through hydrogen bonding with the carbonyl oxygen of Ser<sup>65</sup> to activate the carbonyl carbon for nucleophilic attack by the amide nitrogen of Gly<sup>67</sup>. This mechanism was further supported by ab initio calculations, as well as database searches of similar compounds and protein sequences. Through acid/base chemistry, the chromophore is stabilized by resonance.<ref name="Phillips" /> | <scene name='Green_Fluorescent_Protein/Arg96/1' target='3' >base</scene> by withdrawing electrons through hydrogen bonding with the carbonyl oxygen of Ser<sup>65</sup> to activate the carbonyl carbon for nucleophilic attack by the amide nitrogen of Gly<sup>67</sup>. This mechanism was further supported by ab initio calculations, as well as database searches of similar compounds and protein sequences. Through acid/base chemistry, the chromophore is stabilized by resonance.<ref name="Phillips" /> | ||
===Mutant Studies=== | ===Mutant Studies=== | ||
