A stilbene that binds selectively to transthyretin in cells and remains dark until it undergoes a chemoselective reaction to create a bright blue fluorescent conjugate.
A stilbene that binds selectively to transthyretin in cells and remains dark until it undergoes a chemoselective reaction to create a bright blue fluorescent conjugate.
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DOI：
10.1021/ja104999v
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发表时间：
2010-11-17
影响因子：
15
通讯作者：
Kelly, Jeffery W.
中科院分区：
文献类型：
--
作者：
Choi, Sungwook;Ong, Derrick Sek Tong;Kelly, Jeffery W.
我们描述了一种非荧光的第二代二苯乙烯，它在复杂的生物环境中非常选择性地与甲状腺素运载蛋白结合，并且保持黑暗，直到它与甲状腺素运载蛋白的 pKa 扰动的 Lys-15 ε-氨基发生化学选择性反应，形成亮蓝色荧光缀合物。二苯乙烯 A2 在机制上是不寻常的，因为它在缺乏转甲状腺素蛋白的细胞裂解物中保持非荧光，即使可能存在一些蛋白质组结合。因此，它对于细胞成像特别有用，因为在 A2 与甲状腺素运载蛋白发生反应之前，背景荧光是无法检测到的。报道了 A2 在与转甲状腺素蛋白结合但在反应之前有效缺乏环境敏感荧光的机制基础。 Stilbene A2 在 37 °C 下表现出足够快的转甲状腺素蛋白缀合动力学，能够进行脉冲追踪实验，在本例中证明转甲状腺素蛋白是从 HeLa 细胞中分泌的。作为追逐化合物，我们采用了 C1——一种细胞渗透性、高选择性、非共价、转甲状腺素蛋白结合的二氢芪，不会发出荧光。报告的进展被视为朝着我们创建单链、单结合位点转甲状腺素蛋白标签的长期目标迈出的第一步，也是必要的一步，其荧光可以通过添加 A2 或类似分子来调节。将感兴趣的蛋白质与受 A2 调节的单链、单结合位点运甲状腺素蛋白标签融合，对于利用脉冲追踪实验研究细胞分泌途径中的折叠、运输和降解应该很有用。 A2 的直接应用包括利用其共轭荧光来量化人血浆中转甲状腺素蛋白的浓度，反映营养状况，以及确定动力学稳定剂药物与血浆中转甲状腺素蛋白的结合化学计量。
We describe a non-fluorescent, second generation stilbene that very selectively binds to transthyretin in complex biological environments and remains dark until it chemoselectively reacts with the pKa perturbed Lys-15 ε-amino group of transthyretin to form a bright blue fluorescent conjugate. Stilbene A2 is mechanistically unusual in that it remains non-fluorescent in cell lysates lacking transthyretin, even though there is likely some proteome binding. Thus, it is especially useful for cellular imaging, as background fluorescence is undetectable until A2 reacts with transthyretin. The mechanistic basis for the effective lack of environment-sensitive fluorescence of A2 when bound to, but before reacting with, transthyretin is reported. Stilbene A2 exhibits sufficiently rapid transthyretin conjugation kinetics at 37 °C to enable pulse-chase experiments to be performed, in this case demonstrating that transthyretin is secreted from HeLa cells. As the chase compound, we employed C1–a cell permeable, highly selective, non-covalent, transthyretin binding dihydrostilbene that cannot become fluorescent. The progress reported is viewed as a first and necessary step toward our long-term goal of creating a one-chain, one-binding-site transthyretin tag, whose fluorescence can be regulated by adding A2, or an analogous molecule. Fusing proteins of interest to a one-chain, one-binding-site transthyretin tag regulated by A2 should be useful for studying folding, trafficking and degradation in the cellular secretory pathway, utilizing pulse-chase experiments. Immediate applications of A2 include utilizing its conjugate fluorescence to quantify transthyretin concentration in human plasma, reflecting nutritional status, and determining the binding stoichiometry of kinetic stabilizer drugs to transthyretin in plasma.
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影响因子：
14.8
作者：
Choi S;Connelly S;Reixach N;Wilson IA;Kelly JW
通讯作者：
Kelly JW
影响因子：
48
作者：
Chen, I;Howarth, M;Ting, AY
通讯作者：
Ting, AY
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46.9
作者：
Guignet, EG;Hovius, R;Vogel, H
通讯作者：
Vogel, H
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56.9
作者：
CHALFIE, M;TU, Y;PRASHER, DC
通讯作者：
PRASHER, DC
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56.9
作者：
Debler, Erik W.;Kaufmann, Gunnar F.;Lerner, Richard A.
通讯作者：
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