title: Stabilizing role of glutamic acid 222 in the structure of Enhanced Green Fluorescent Protein.
abstract: Enhanced Green Fluorescent Protein (EGFP) is a variant of wild-type Green Fluorescent Protein from the jellyfish Aequorea victoria, whose mutations S65T and F64L increase brightness and folding efficiency. EGFP is extensively used in cell biology and biochemistry as a colocalization or expression reporter. Surprisingly, the structure of this very popular protein has not been determined yet. We report here its crystallographic structure at 1.5Å resolution which shows significant differences in the vicinity of residue 64 and of the chromophore. In particular, two conformations are observed for the key residue glutamic acid 222, in apparent contradiction with the single fluorescence lifetime of the protein. We then show that X-ray induced decarboxylation of Glu222 during diffraction data collection results in the disruption of a hydrogen-bond network near the chromophore. Using single-crystal microspectrophotometry, we demonstrate that this correlates with a significant loss of the fluorescence properties. We thus propose a mechanism of bleaching of the protein at low temperature. Taken together, these two sets of results highlight the stabilizing role of Glu222 to the chromophore cavity of EGFP.
authors.affiliation: CNRS, Institut de Biologie Structurale Jean-Pierre Ebel, 41 rue Jules Horowitz, 38027 Grenoble, France. antoine.royant@ibs.fr
authors.forename: Antoine
authors.initials: A
authors.lastname: Royant
authors.forename: Marjolaine
authors.initials: M
authors.lastname: Noirclerc-Savoye
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D005453:Fluorescence; D018698:Glutamic Acid / Q000737:chemistry* / Q000528:radiation effects; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D006829:Hydra; D009154:Mutation; D055550:Protein Stability; D017434:Protein Structure, Tertiary
chemical_list: D049452:Green Fluorescent Proteins; D018698:Glutamic Acid