Significant expansion and red-shifting of fluorescent protein chromophore determined through computational design and genetic code expansion  

作　　者：Li Wang Xian Chen Xuzhen GUO liasong Li Qi Liu Fuying Kang Xudong Wang Cheng HU Haiping Liu Weimin Gong Wei Zhuang Xiaohong Liu Jiangyun Wang 

机构地区：[1]Institute of Biophysics,Chinese Academy of Sciences,Beijing 100101,China [2]College of Life Sciences,University of Chinese Academy of Sciences,Beijing 100049,China [3]Key Laboratory of Physics and Technology for Advanced Batteries (Ministry of Education),Department of Physics,Jilin University,Changchun 130012,China [4]State Key Laboratory of Structural Chemistry,Fujian Institute of Research on the Structure of Matter,Chinese Academy of Sciences,Fuzhou 350002,China

出　　处：《Biophysics Reports》2018年第5期273-285,共13页生物物理学报（英文版）

基　　金：This work was financially supported in part by Grants from the National Natural Science Foundation of China (21750003,91527302,U1632133,31628004,21473237,and 31628004);the National Key Research and Development Program of China (2017YFA0503704and 2016YFA0501502);and the Key Research Program of Frontier Sciences,CAS(QYZDB-SSW- SMC032).

摘　　要：Fluorescent proteins(FPs) with emission wavelengths in the far-red and infrared regions of the spectrum provide powerful tools for deep-tissue and super-resolution imaging. The development of red-shifted FPs has evoked widespread interest and continuous engineering efforts. In this article,based on a computational design and genetic code expansion, we report a rational approach to significantly expand and red-shift the chromophore of green fluorescent protein(GFP). We applied computational calculations to predict the excitation and emission wavelengths of a FP chromophore harboring unnatural amino acids(UAA) and identify in silico an appropriate UAA, 2-amino-3-(6-hydroxynaphthalen-2-yl)propanoic acid(naphthol-Ala). Our methodology allowed us to formulate a GFP variant(cpsf GFP-66-Naphthol-Ala) with red-shifted absorbance and emission spectral maxima exceeding 60 and 130 nm, respectively, compared to those of GFP. The GFP chromophore is formed through autocatalytic post-translational modification to generate a planar 4-(p-hydroxybenzylidene)-5-imidazolinone chromophore. We solved the crystal structure of cpsf GFP-66-naphthol-Ala at 1.3 ? resolution and demonstrated the formation of a much larger conjugated p-system when the phenol group is replaced by naphthol. These results explain the significant red-shifting of the excitation and emission spectra of cpsf GFP-66-naphthol-Ala.

分 类 号：Q[生物学]