Random single amino acid deletion sampling unveils structural tolerance and the benefits of helical registry shift on GFP folding and structure.
Random single amino acid deletion sampling unveils structural tolerance and the benefits of helical registry shift on GFP folding and structure.
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DOI：
10.1016/j.str.2014.03.014
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发表时间：
2014-06-10
期刊：
影响因子：
--
通讯作者：
Jones DD
中科院分区：
文献类型：
--
作者：
Arpino JA;Reddington SC;Halliwell LM;Rizkallah PJ;Jones DD
通过氨基酸缺失改变蛋白质骨架是一种常见的进化突变机制，但在蛋白质工程中被普遍忽视，主要是因为它对折叠-结构-功能关系的影响难以预测。利用定向进化，观察到增强型绿色荧光蛋白(EGFP)可以容忍整个蛋白质范围内的残基缺失，特别是在短环和长环、螺旋元件和链的末端。从螺旋上去除G4的变异体(EGFPG4Δ)具有显著更高的细胞荧光。折叠分析表明，EGFPG4Δ在去折叠时保留了更多的结构，复性效率几乎为100%，但以热力学稳定性为代价。G4的Δ结构表明，G4的缺失导致了有益的螺旋注册表移动，导致了一个新的极性相互作用网络，该网络可能稳定顺式脯氨酸肽键并连接二级结构元素。因此，缺失突变和注册表移位可以通过结构重排来增强蛋白质，而单靠替换突变是不可能的。利用定向进化，研究了氨基酸缺失对EGFP的影响。环、螺旋和链末端对氨基酸缺失特别耐受。Arpino等人的结构表明，螺旋注册表移位使用定向进化创建了一个新的极性网络。研究氨基酸缺失对EGFP的影响，发现环、螺旋和链末端对氨基酸缺失特别耐受。结构工作为这一观察到的现象提供了分子解释。
Altering a protein’s backbone through amino acid deletion is a common evolutionary mutational mechanism, but is generally ignored during protein engineering primarily because its effect on the folding-structure-function relationship is difficult to predict. Using directed evolution, enhanced green fluorescent protein (EGFP) was observed to tolerate residue deletion across the breadth of the protein, particularly within short and long loops, helical elements, and at the termini of strands. A variant with G4 removed from a helix (EGFPG4Δ) conferred significantly higher cellular fluorescence. Folding analysis revealed that EGFPG4Δ retained more structure upon unfolding and refolded with almost 100% efficiency but at the expense of thermodynamic stability. The EGFPG4Δ structure revealed that G4 deletion caused a beneficial helical registry shift resulting in a new polar interaction network, which potentially stabilizes a cis proline peptide bond and links secondary structure elements. Thus, deletion mutations and registry shifts can enhance proteins through structural rearrangements not possible by substitution mutations alone. Using directed evolution, the impact of amino acid deletion on EGFP is explored Loops, helices, and strand termini are especially tolerant to amino acid deletion A deletion mutant that enhances cellular production and fluorescence is identified Structure reveals that a helical registry shift creates a new polar network Using directed evolution, Arpino et al. examine the impact of amino acid deletion on EGFP and find that loops, helices, and strand termini are especially tolerant to amino acid deletion. Structural work provides a molecular explanation for this observation.
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DOI：
10.1107/s0907444905036693
发表时间：
2006-01-01
影响因子：
2.2
作者：
Evans, P
通讯作者：
Evans, P
DOI：
10.1098/rspb.2011.2571
发表时间：
2012-08-07
期刊：
Proceedings. Biological sciences
影响因子：
--
作者：
Leushkin EV;Bazykin GA;Kondrashov AS
通讯作者：
Kondrashov AS
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.7
作者：
Arpino JA;Rizkallah PJ;Jones DD
通讯作者：
Jones DD
影响因子：
2.9
作者：
Enoki, S;Saeki, K;Kuwajima, K
通讯作者：
Kuwajima, K