Directed evolution of GFP with non-natural amino acids identifies residues for augmenting and photoswitching fluorescence.
Directed evolution of GFP with non-natural amino acids identifies residues for augmenting and photoswitching fluorescence.
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DOI：
10.1039/c4sc02827a
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发表时间：
2015-02-01
期刊：
影响因子：
8.4
通讯作者：
Jones DD
中科院分区：
文献类型：
--
作者：
Reddington SC;Baldwin AJ;Thompson R;Brancale A;Tippmann EM;Jones DD
遗传密码重新编程允许蛋白质通过有针对性地引入非天然氨基酸来采样新的化学物质。通过与随机密码子替换相结合，传统上被忽视的残基可以被识别为向目标蛋白质灌输新的性质。遗传密码重新编程允许蛋白质通过明确和有针对性地引入非天然氨基酸(NAA)来采样新的化学物质。许多有用的NaA是天然芳香氨基酸酪氨酸的衍生物，对羟基被有用但通常更大的取代基取代。通过定向进化扩展残基采样，确定了绿色荧光蛋白中对芳香族NaAs耐受的位置，包括鉴定了调节荧光的新位点。用光敏的对叠氮苯丙氨酸(AZF)取代埋藏的L44残基，实现了环境敏感的光开关。在电子计算机模拟中，L44azF的暗态通过调节生色团周围的氢键网络提供了对作用机制的洞察。将T203与芳香族NaA定向突变，引入π堆积，成功地产生了红移版本的绿色荧光蛋白。在203位残基上掺入AZF使sfGFP具有很高的光敏性，甚至环境光也能调节功能开关。因此，通过研究广泛的残基来设计含有非天然芳香族氨基酸的工程蛋白质，可以通过采样非直觉突变为蛋白质引入新的和有益的性质。再加上电子计算机模型的回顾，这将有助于我们了解NaAS对蛋白质结构和功能的影响，以及未来的设计努力。
Genetic code reprogramming allows proteins to sample new chemistry through targeted introduction of non-natural amino acids. By combining with random codon replacement, residues traditionally overlooked can be identified as instilling new properties on a target protein. Genetic code reprogramming allows proteins to sample new chemistry through the defined and targeted introduction of non-natural amino acids (nAAs). Many useful nAAs are derivatives of the natural aromatic amino acid tyrosine, with the para OH group replaced with useful but often bulkier substituents. Extending residue sampling by directed evolution identified positions in Green Fluorescent Protein tolerant to aromatic nAAs, including identification of novel sites that modulate fluorescence. Replacement of the buried L44 residue by photosensitive p-azidophenylalanine (azF) conferred environmentally sensitive photoswitching. In silico modelling of the L44azF dark state provided an insight into the mechanism of action through modulation of the hydrogen bonding network surrounding the chromophore. Targeted mutagenesis of T203 with aromatic nAAs to introduce π-stacking with the chromophore successfully generated red shifted versions of GFP. Incorporation of azF at residue 203 conferred high photosensitivity on sfGFP with even ambient light mediating a functional switch. Thus, engineering proteins with non-natural aromatic amino acids by surveying a wide residue set can introduce new and beneficial properties into a protein through the sampling of non-intuitive mutations. Coupled with retrospective in silico modelling, this will facilitate both our understanding of the impact of nAAs on protein structure and function, and future design endeavours.
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影响因子：
4.9
作者：
Reddington, Samuel C.;Tippmann, Eric M.;Jones, D. Dafydd
通讯作者：
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