Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein.
Structural and dynamic changes associated with beneficial engineered single-amino-acid deletion mutations in enhanced green fluorescent protein.
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DOI：
10.1107/s139900471401267x
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发表时间：
2014-08
期刊：
影响因子：
--
通讯作者：
Jones DD
中科院分区：
文献类型：
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作者：
Arpino JA;Rizkallah PJ;Jones DD
已经研究了有益的工程化单氨基酸缺失变体EGFPD 190 Δ和EGFPA 227 Δ。单氨基酸缺失是自然进化景观的常见部分，但由于对缩短蛋白质骨架的突变的有限和偏见的分子理解，在蛋白质工程中很少取样。增强型绿色荧光蛋白（EGFP）的单氨基酸缺失变体已经通过定向进化鉴定，其具有赋予增加的细胞荧光的有益效果。生物物理特性表明，增加功能蛋白质的生产，而不是改变荧光参数的机制，可能是负责。在环内含有缺失的结构EGFPD 190 Δ仅在缺失的残基之后显示出传播性变化。EGFPA227Δ的结构显示，使用“翻转”机制来调整β链末端的残基缺失，缺失位点C端的氨基酸重新定位以取代缺失的氨基酸。在这两种变体中，产生了新的短程和长程相互作用网络，同时保持了疏水核心的完整性。这两种缺失变体也显示出显着的本地和远程的动态变化，明显的B因子与EGFP相比的变化。缺失突变不是有害的，而是可以通过改变核心蛋白的性质、折叠和动力学以及功能来引入有益的结构效应。
The beneficial engineered single-amino-acid deletion variants EGFPD190Δ and EGFPA227Δ have been studied. Single-amino-acid deletions are a common part of the natural evolutionary landscape but are rarely sampled during protein engineering owing to limited and prejudiced molecular understanding of mutations that shorten the protein backbone. Single-amino-acid deletion variants of enhanced green fluorescent protein (EGFP) have been identified by directed evolution with the beneficial effect of imparting increased cellular fluorescence. Biophysical characterization revealed that increased functional protein production and not changes to the fluorescence parameters was the mechanism that was likely to be responsible. The structure EGFPD190Δ containing a deletion within a loop revealed propagated changes only after the deleted residue. The structure of EGFPA227Δ revealed that a ‘flipping’ mechanism was used to adjust for residue deletion at the end of a β-strand, with amino acids C-terminal to the deletion site repositioning to take the place of the deleted amino acid. In both variants new networks of short-range and long-range interactions are generated while maintaining the integrity of the hydrophobic core. Both deletion variants also displayed significant local and long-range changes in dynamics, as evident by changes in B factors compared with EGFP. Rather than being detrimental, deletion mutations can introduce beneficial structural effects through altering core protein properties, folding and dynamics, as well as function.