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

Li Wang,Jiasong Li,Jiangyun Wang,Fuying Kang,Xian Chen,Cheng Hu,Wei Zhuang,Qi Liu,Xuzhen Guo,Haiping Liu,Weimin Gong,Xiaohong Liu,Xudong Wang

doi:10.1007/s41048-018-0073-z

Abstract

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 (cpsfGFP-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 cpsfGFP-66-naphthol-Ala at 1.3 Å resolution and demonstrated the formation of a much larger conjugated π-system when the phenol group is replaced by naphthol. These results explain the significant red-shifting of the excitation and emission spectra of cpsfGFP-66-naphthol-Ala.Graphical abstract

Highlights

Fluorescent proteins (FPs) with far-red and infraredregion emission wavelengths are powerful tools for tracking protein localization in cells, cell migration, and deep-tissue imaging (Enterina et al 2015; Nienhaus and Nienhaus 2014; Niu and Guo 2013; Sengupta et al 2014; Shaner et al 2011)
When the 66Tyr residue of the chromophore in various FPs is substituted with HqAla, excitation and emission spectra red-shifted by 30 nm are achieved (Liu et al 2013)
When the sfGFP 66Tyr site is substituted with a heterocyclic amino acid, the resulting excitation and emission spectra are red-shifted by approximately 30 nm (Liu et al 2013)

Summary

Introduction

Fluorescent proteins (FPs) with far-red and infraredregion emission wavelengths are powerful tools for tracking protein localization in cells, cell migration, and deep-tissue imaging (Enterina et al 2015; Nienhaus and Nienhaus 2014; Niu and Guo 2013; Sengupta et al 2014; Shaner et al 2011). CpsfGFP-66-naphthol-Ala has significantly red-shifted absorbance and emission spectral maxima compared to those of wild-type green FP (GFP). The oxidation red fluorescence of cpsfGFP-66-Naphthol-Ala revealed remarkably red-shifted excitation and emission spectra.

Results

Conclusion