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

• 来源期刊: 生物物理学报
• 年，卷（期）: 2018,（5）
• 页码范围: 273-285
• 页数: 13页
• 语种: 中文

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 (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 (A) 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.