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Structural analysis of the bright monomeric yellow‐green fluorescent protein mNeonGreen obtained by directed evolution
Author(s) -
Clavel Damien,
Gotthard Guillaume,
von Stetten David,
De Sanctis Daniele,
Pasquier Hélène,
Lambert Gerard G.,
Shaner Nathan C.,
Royant Antoine
Publication year - 2016
Publication title -
acta crystallographica section d
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.374
H-Index - 138
ISSN - 2059-7983
DOI - 10.1107/s2059798316018623
Subject(s) - green fluorescent protein , fluorescence , chromophore , chemistry , monomer , fluorescent protein , gene , biophysics , biology , photochemistry , biochemistry , optics , physics , organic chemistry , polymer
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 lan YFP from Branchiostoma lanceolatum . A successful monomerization attempt led to the development of the bright yellow‐green fluorescent protein mNeonGreen. The structures of lan YFP 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.