Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%.
Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%.
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DOI：
10.1038/ncomms1738
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发表时间：
2012-03-20
影响因子：
16.6
通讯作者：
中科院分区：
文献类型：
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作者：
绿色荧光蛋白的青色变体被广泛用作福斯特共振能量转移实验中的供体。流行的，但适度明亮，增强型青色荧光蛋白（ECFP）依次改进为更明亮的变体超级青色荧光蛋白3A（SCFP3A）和mTurquoise，后者表现出高荧光量子产率和长单指数荧光寿命。在这里，我们结合联合收割机X射线晶体学和激发态计算合理化这些逐步改善。这种增强源于第七条β链的稳定和唯一的发色团稳定氢键的加强。结构分析突出了一个次优的内部残基，其经受饱和诱变结合基于荧光寿命的筛选。这导致了mTurquoise 2，一种更明亮的变体，具有更快的成熟，高光稳定性，更长的单指数寿命和单体荧光蛋白测量的最高量子产率。总之，这些特性使mTurquoise 2成为长期成像的绿色荧光蛋白的优选青色变体，并作为Förster共振能量转移到黄色荧光蛋白的供体。绿色荧光蛋白（CFPs）的青色变体被广泛用作FRET实验中的供体。在这里，开发了一种新的CFP，mTurquoise2，它显示出高荧光量子产率和长的单指数荧光寿命。
Cyan variants of green fluorescent protein are widely used as donors in Förster resonance energy transfer experiments. The popular, but modestly bright, Enhanced Cyan Fluorescent Protein (ECFP) was sequentially improved into the brighter variants Super Cyan Fluorescent Protein 3A (SCFP3A) and mTurquoise, the latter exhibiting a high-fluorescence quantum yield and a long mono-exponential fluorescence lifetime. Here we combine X-ray crystallography and excited-state calculations to rationalize these stepwise improvements. The enhancement originates from stabilization of the seventh β-strand and the strengthening of the sole chromophore-stabilizing hydrogen bond. The structural analysis highlighted one suboptimal internal residue, which was subjected to saturation mutagenesis combined with fluorescence lifetime-based screening. This resulted in mTurquoise2, a brighter variant with faster maturation, high photostability, longer mono-exponential lifetime and the highest quantum yield measured for a monomeric fluorescent protein. Together, these properties make mTurquoise2 the preferable cyan variant of green fluorescent protein for long-term imaging and as donor for Förster resonance energy transfer to a yellow fluorescent protein. Cyan variants of green fluorescent protein (CFPs) are widely used as donors in FRET experiments. Here, a new CFP, mTurquoise2, is developed, which displays a high-fluorescence quantum yield and a long mono-exponential fluorescence lifetime.
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影响因子：
3.7
作者：
Alieva NO;Konzen KA;Field SF;Meleshkevitch EA;Hunt ME;Beltran-Ramirez V;Miller DJ;Wiedenmann J;Salih A;Matz MV
通讯作者：
Matz MV
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：
46.9
作者：
Nagai, T;Ibata, K;Miyawaki, A
通讯作者：
Miyawaki, A
DOI：
10.1107/s0567740871004060
发表时间：
1971-01-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION B-STRUCTURAL CRYSTALLOGRAPHY AND CRYSTAL CHEMISTRY
影响因子：
--
作者：
HENDERSON, R;MOFFAT, JK
通讯作者：
MOFFAT, JK
影响因子：
2.9
作者：
Kremers, Gert-Jan;Goedhart, Joachim;Gadella, Theodorus W. J., Jr.
通讯作者：
Gadella, Theodorus W. J., Jr.