Crystal structure of enhanced green fluorescent protein to 1.35 Å resolution reveals alternative conformations for Glu222.
Crystal structure of enhanced green fluorescent protein to 1.35 Å resolution reveals alternative conformations for Glu222.
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DOI：
10.1371/journal.pone.0047132
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Jones DD
中科院分区：
文献类型：
--
作者：
Arpino JA;Rizkallah PJ;Jones DD
增强型绿色荧光蛋白 (EGFP) 是原始野生型绿色荧光蛋白的最广泛使用的工程变体之一。在这里，我们报告了以未标记序列形式结晶的 EGFP 的高分辨率 (1.35 Å) 结构，揭示了 F64L 和 S65T 的综合影响，从而改善了折叠和光谱特性。 EGFP的整体结构与wt GFP非常相似，形成经典的β-桶状折叠，其中含有发色团的螺旋贯穿结构的核心。在 EGFP 中用 Leu 替换 Phe64 会导致靠近发色团的疏水核心堆积发生微妙的重排，包括减少两个疏水残基的表面暴露。用 Thr 替换 Ser65 对发色团附近的局部氢键网络有显着影响。电子密度的详细分析表明，靠近发色团的几个残基占据至少两种不同的构象。其中包括定义发色团带电状态的 Glu222，这两种构象对发色团周围的氢键网络的影响略有不同。因此，报道的 EGFP 高分辨率结构为目前普遍使用的最重要的荧光蛋白变体之一提供了早该进行的分子描述。
Enhanced Green Fluorescent Protein (EGFP) is one of the most widely used engineered variants of the original wild-type Green Fluorescent Protein. Here, we report the high resolution (1.35 Å) structure of EGFP crystallised in its untagged sequence form that reveals the combined impact of the F64L and S65T, that give rise to improved folding and spectral characteristics. The overall structure of EGFP is very similar to wt GFP, forming the classical β-barrel fold with the chromophore containing helix running through the core of the structure. Replacement of Phe64 with Leu in EGFP results in subtle rearrangement of hydrophobic core packing close to the chromophore including the reduction in surface exposure of two hydrophobic residues. Replacement of Ser65 with Thr has a significant impact on the local hydrogen bond network in the vicinity of the chromophore. Detailed analysis of electron density reveals that several residues close to the chromophore occupy at least two distinct conformations. This includes Glu222 that defines the charged state on the chromophore, with the two conformations having slightly different effects on the hydrogen bond network surrounding the chromophore. Hence, the reported high-resolution structure of EGFP has provided a long overdue molecular description of one of the most important fluorescent protein variants currently in general use.
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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
影响因子：
3.4
作者：
Patterson, GH;Knobel, SM;Piston, DW
通讯作者：
Piston, DW
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：
3.5
作者：
Cormack, BP;Valdivia, RH;Falkow, S
通讯作者：
Falkow, S
影响因子：
--
作者：
SHIMOMURA, O;JOHNSON, FH;SAIGA, Y
通讯作者：
SAIGA, Y