Structural analysis of the bright monomeric yellow-green fluorescent protein mNeonGreen obtained by directed evolution

• Published in: Acta crystallographica. Section D, Biological crystallography. Vol. 72; no. Pt 12; p. 1298
• Main Authors: Clavel, Damien, Gotthard, Guillaume, von Stetten, David, De Sanctis, Daniele, Pasquier, Hélène, Lambert, Gerard G, Shaner, Nathan C, Royant, Antoine
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.12.2016
• Subjects: Animals; Cloning, Molecular; Crystallography, X-Ray; Directed Molecular Evolution - methods; Genes; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Lancelets - chemistry; Lancelets - genetics; Luminescent Proteins - chemistry; Luminescent Proteins - genetics; Models, Molecular; Mutation; Protein Conformation; Proteins; Spectrum Analysis, Raman; Branchiostoma lanceolatum; fluorescent protein; online Raman spectroscopy; specific radiation damage; in crystallo optical spectroscopy
• ISSN: 0907-4449; 2059-7983; 1399-0047
• DOI: 10.1107/s2059798316018623

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.