Green Fluorescent Protein: Difference between revisions

Three amino residues in the central α-helix constitute the fluorophore of GFP: Ser⁶⁵Tyr⁶⁶Gly⁶⁷ (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 Ser⁶⁵ and Gly⁶⁷ to form an imidazolin-5-one intermediate.<ref name="Yang" />  This rapid cyclization is carried out via nucleophilic attack of the amino group from Gly⁶⁷ on the carbonyl group of Ser⁶⁵ 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 Tyr⁶⁶ 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>

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" />