Genetically Encoding Quinoline Reverses Chromophore Charge and Enables Fluorescent Protein Brightening in Acidic Vesicles.
Genetically Encoding Quinoline Reverses Chromophore Charge and Enables Fluorescent Protein Brightening in Acidic Vesicles.
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DOI：
10.1021/jacs.8b05814
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发表时间：
2018-09-05
影响因子：
15
通讯作者：
Wang L
中科院分区：
文献类型：
--
作者：
Fu C;Kobayashi T;Wang N;Hoppmann C;Yang B;Irannejad R;Wang L
酸性囊泡和细胞器在广泛的细胞事件中发挥重要作用，如内吞作用、溶酶体降解、突触传递、病原体命运和药物递送。荧光报告分子对于研究这些具有时空分辨率的复杂和多功能系统将是非常宝贵的，然而常见的荧光蛋白在酸性条件下由于质子化后阴离子发色团的减少而通常是非荧光的，但是在生理pH下是荧光的，从而产生来自非囊泡区域的干扰荧光。在这里，我们开发了一种新的abFP（酸性增亮荧光蛋白），其在酸性pH下发出强烈荧光，但在中性pH或高于中性pH下不发荧光，其pH曲线与普通荧光蛋白相反。通过扩展遗传密码，我们将含喹啉的氨基酸Qui掺入到EGFP的发色团中以逆转发色团电荷。Qui的质子化使其成为一个阳离子发色团，在体外仅在酸性pH下才能产生独特的荧光增强。coli细胞和哺乳动物细胞表面。我们进一步证明，abFP标记的δ阿片受体在溶酶体中荧光成像，显示出不同的特征，并且没有来自其他细胞区域的背景荧光，而EGFP标记的受体在溶酶体中不可见。这种Qui呈现的阳离子发色团策略通常可以应用于其他荧光蛋白，以产生用于具有最小背景的酸性成像的调色板，并且这些abFP应该促进与不同细胞和模式生物中的各种酸性囊泡和细胞器相关的分子的研究。
Acidic vesicles and organelles play fundamental roles in a broad range of cellular events such as endocytosis, lysosomal degradation, synaptic transmission, pathogen fate, and drug delivery. Fluorescent reporters will be invaluable for studying these complex and multifunctional systems with spatiotemporal resolution, yet common fluorescent proteins are generally non-fluorescent at acidic conditions due to the decrease of anionic chromophore upon protonation, but are fluorescent at physiological pH creating interfering fluorescence from non-vesicle regions. Here we developed a novel abFP (acid brightening fluorescent protein) that fluoresces strongly at acidic pH but is non-fluorescent at or above neutral pH, boasting a pH profile opposite to that of common fluorescent proteins. Through expansion of the genetic code, we incorporated a quinoline-containing amino acid Qui into the chromophore of EGFP to reverse the chromophore charge. Protonation of Qui rendered a cationic chromophore, which resulted in unique fluorescence increase only at acidic pH in vitro, in E. coli cells, and on mammalian cell surface. We further demonstrated that abFP tagged δ opioid receptors were fluorescently imaged in lysosome showing distinct features and without background fluorescence from other cellular regions, whereas EGFP tagged receptors were invisible in lysosome. This Qui-rendered cationic chromophore strategy may be generally applied to other fluorescent proteins to generate a palette of colors for acidic imaging with minimal background, and these abFPs should facilitate the study of molecules in association with various acidic vesicles and organelles in different cells and model organisms.
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影响因子：
3.2
作者：
Lacey, Vanessa K.;Louie, Gordon V.;Noel, Joseph P.;Wang, Lei
通讯作者：
Wang, Lei
DOI：
10.1038/nrm3565
发表时间：
2013-05
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
通讯作者：
--
影响因子：
14.8
作者：
Ferrandon, Sebastien;Feinstein, Timothy N.;Castro, Marian;Wang, Bin;Bouley, Richard;Potts, John T.;Gardella, Thomas J.;Vilardaga, Jean-Pierre
通讯作者：
Vilardaga, Jean-Pierre
影响因子：
16.2
作者：
Kang JY;Kawaguchi D;Coin I;Xiang Z;O'Leary DD;Slesinger PA;Wang L
通讯作者：
Wang L
影响因子：
9.8
作者：
Calebiro D;Nikolaev VO;Gagliani MC;de Filippis T;Dees C;Tacchetti C;Persani L;Lohse MJ
通讯作者：
Lohse MJ