title: Structural analysis of the bright monomeric yellow-green fluorescent protein mNeonGreen obtained by directed evolution.
abstract: Until recently, genes coding for homologues of the autofluorescent protein GFP had only been identified in marine organisms from the phyla Cnidaria and Arthropoda. New fluorescent-protein genes have now been found in the phylum Chordata, coding for particularly bright oligomeric fluorescent proteins such as the tetrameric yellow fluorescent protein lanYFP from Branchiostoma lanceolatum. A successful monomerization attempt led to the development of the bright yellow-green fluorescent protein mNeonGreen. The structures of lanYFP and mNeonGreen have been determined and compared in order to rationalize the directed evolution process leading from a bright, tetrameric to a still bright, monomeric fluorescent protein. An unusual discolouration of crystals of mNeonGreen was observed after X-ray data collection, which was investigated using a combination of X-ray crystallography and UV-visible absorption and Raman spectroscopies, revealing the effects of specific radiation damage in the chromophore cavity. It is shown that X-rays rapidly lead to the protonation of the phenolate O atom of the chromophore and to the loss of its planarity at the methylene bridge.
authors.affiliation: Université Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38044 Grenoble, France.
authors.forename: Damien
authors.initials: D
authors.lastname: Clavel
authors.affiliation: European Synchrotron Radiation Facility, F-38043 Grenoble, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Gotthard
authors.affiliation: European Synchrotron Radiation Facility, F-38043 Grenoble, France.
authors.forename: David
authors.initials: D
authors.lastname: von Stetten
authors.affiliation: European Synchrotron Radiation Facility, F-38043 Grenoble, France.
authors.forename: Daniele
authors.initials: D
authors.lastname: De Sanctis
authors.affiliation: CNRS, Laboratoire de Chimie Physique, UMR 8000 and Université Paris-Sud, F-91405 Orsay, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Pasquier
authors.affiliation: Department of Photobiology and Bioimaging, The Scintillon Institute, San Diego, California, USA.
authors.forename: Gerard G
authors.initials: GG
authors.lastname: Lambert
authors.affiliation: Department of Photobiology and Bioimaging, The Scintillon Institute, San Diego, California, USA.
authors.forename: Nathan C
authors.initials: NC
authors.lastname: Shaner
authors.affiliation: Université Grenoble Alpes, Institut de Biologie Structurale (IBS), F-38044 Grenoble, France.
authors.forename: Antoine
authors.initials: A
authors.lastname: Royant
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution / Q000379:methods; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D064175:Lancelets / Q000737:chemistry* / Q000235:genetics; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins
keywords: Branchiostoma lanceolatum; fluorescent protein; in crystallo optical spectroscopy; online Raman spectroscopy; specific radiation damage