Green Fluorescent Protein: Difference between revisions
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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" /> | 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" /> | ||
<applet load='1ema' size='500' frame='true' align='right' scene='Green_Fluorescent_Protein/Polar_interactions/ | <applet load='1ema' size='500' frame='true' align='right' scene='Green_Fluorescent_Protein/Polar_interactions/2' name='2'/> | ||
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">PMID: 17040991</ref> | 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">PMID: 17040991</ref> | ||
The opposite side of the chromophore, however, is within close proximity of several aromatic and polar side chains. Several <scene name='Green_Fluorescent_Protein/Polar_interactions/ | The opposite side of the chromophore, however, is within close proximity of several aromatic and polar side chains. Several <scene name='Green_Fluorescent_Protein/Polar_interactions/2'>polar interactions</scene> between the surrounding residues and the chromophore are present including: hydrogen bonds of His<sup>148</sup>, Thr<sup>203</sup>, and Ser<sup>205</sup> with the phenolic hydroxyl of Tyr<sup>66</sup>; Arg<sup>96</sup> and Gln<sup>94</sup> with the carbonyl of the imidazolidinone ring; and hydrogen bonds of Glu<sup>222</sup> with the side chain of Thr<sup>65</sup>. Additional hydrogen bonding in the area around the chromophore helps to stabilize Arg<sup>96</sup> in the protonated form, which suggests the presence of a partial negative charge on the carbonyl oxygen of the imidazolidinone ring in the deprotonated fluorophore.<ref name="Ormo" /> Arg<sup>96</sup> and Gln<sup>94</sup> in turn help to steady the imidazolidone.<ref name="Yang" /> Therefore, it is thought that Arg<sup>96</sup> is essential for the formation of the fluorophore by catalyzing the initial ring closure.<ref name="Ormo" /> Tyr<sup>145</sup> provides a stabilizing | ||
<scene name='Green_Fluorescent_Protein/Edge_face_interaction/1'>edge-face interaction</scene> with the benzyl ring of the chromophore.<ref name="Ormo" /> (For more information about edge-face (CH/π) interactions, click [http://www.tim.hi-ho.ne.jp/dionisio/ here].) The stability provided by the internal polar interactions are further augmented by the surrounding β-barrel. | <scene name='Green_Fluorescent_Protein/Edge_face_interaction/1'>edge-face interaction</scene> with the benzyl ring of the chromophore.<ref name="Ormo" /> (For more information about edge-face (CH/π) interactions, click [http://www.tim.hi-ho.ne.jp/dionisio/ here].) The stability provided by the internal polar interactions are further augmented by the surrounding β-barrel. | ||
