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

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• 作者：Damien Clavel ; Guillaume Gotthard ; David von Stetten ; Daniele De Sanctis ; Hé ; lè ; ne Pasquier ; Gerard G. Lambert ; Nathan C. Shaner and Antoine Royant
• 关键词：fluorescent protein ; Branchiostoma lanceolatum ; specific radiation damage ; in crystallo optical spectroscopy ; online Raman spectroscopy
• 刊名：Acta Crystallographica Section D
• 出版年：2016
• 出版时间：December 2016
• 年：2016
• 卷：72
• 期：12
• 页码：1298-1307
• 全文大小：1936K
• ISSN：1399-0047

文摘

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.