An in vivo covalent TMP-tag based on proximity-induced reactivity.
An in vivo covalent TMP-tag based on proximity-induced reactivity.
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DOI：
10.1021/cb900062k
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发表时间：
2009-07-17
影响因子：
4
通讯作者：
Cornish, Virginia W.
中科院分区：
文献类型：
--
作者：
Gallagher, Sarah S.;Sable, Julia E.;Sheetz, Michael P.;Cornish, Virginia W.
用于活细胞成像的化学标签正在成为荧光蛋白的可行替代品，用于用小分子探针标记蛋白质。在已报道的化学标签中，甲氧苄啶 (TMP) 标签因具有足够的细胞渗透性和选择性以允许对细胞内蛋白质进行成像而脱颖而出。 TMP 标签提供了一种非共价标记，其中目标蛋白与大肠杆菌二氢叶酸还原酶 (DHFR) 融合，然后用细胞可渗透的 TMP 探针异二聚体进行标记。为了补充非共价 TMP 标签的实用性，我们试图使 TMP 标签共价，用于单分子跟踪和脉冲追踪标记等应用，这些应用将受益于更持久的修饰。基于邻近诱导反应性在不可逆抑制剂设计中的长期使用及其最近在体外化学生物学工具中的应用，我们设计了一种 eDHFR 变体，其具有独特的 Cys 残基，可与安装在 TMP 探针标签上的丙烯酰胺亲电子试剂发生反应。体外实验表明，eDHFR:Leu28Cys 亲核试剂与 TMP-丙烯酰胺探针快速定量反应。最重要的是，丙烯酰胺亲电子试剂提供的反应性平衡使得用 eDHFR:Leu28Cys 标记的细胞内蛋白能够在活细胞中用 TMP-丙烯酰胺-荧光素异三聚体进行标记，背景最小。因此，此处描述的 TMP 亲电子试剂可立即用作活细胞中的共价化学标签。此外，接近诱导的反应性被证明对于在活细胞中使用具有足够的选择性，这表明从现有的非共价配体-蛋白质对开发正交共价化学标签的通用方法。
Chemical tags for live cell imaging are emerging as viable alternatives to the fluorescent proteins for labeling proteins with small molecule probes. Among reported chemical tags, trimethoprim (TMP)-tag stands out for having sufficient cell permeability and selectivity to allow imaging of intracellular proteins. TMP-tag provides a non-covalent label in which the protein of interest is fused to E. coli dihydrofolate reductase (DHFR) and then labeled with a cell permeable TMP-probe heterodimer. To complement the utility of the non-covalent TMP-tag, we sought to render the TMP-tag covalent for applications such as single-molecule tracking and pulse-chase labeling that would benefit from a more permanent modification. Based on the long-standing use of proximity-induced reactivity for irreversible inhibitor design and its more recent application to in vitro chemical biology tools, we designed an eDHFR variant with a unique Cys residue positioned to react with an acrylamide electrophile installed on the TMP-probe label. In vitro experiments show that the eDHFR:Leu28Cys nucleophile reacts rapidly and quantitatively with the TMP-acrylamide-probe. Most significantly, the balance in reactivity provided by the acrylamide electrophile allows intracellular proteins tagged with eDHFR:Leu28Cys to be labeled with a TMP-acrylamide-fluorescein heterotrimer in live cells with minimal background. Thus, the TMP electrophile described here can be used immediately as a covalent chemical tag in live cells. Moreover, proximity-induced reactivity is shown to be sufficiently selective for use in a living cell, suggesting a general approach for the development of orthogonal covalent chemical tags from existing non-covalent ligand-protein pairs.
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影响因子：
48
作者：
Chen, I;Howarth, M;Ting, AY
通讯作者：
Ting, AY
影响因子：
56.9
作者：
Bates, Mark;Huang, Bo;Zhuang, Xiaowei
通讯作者：
Zhuang, Xiaowei
DOI：
10.1073/pnas.151260298
发表时间：
2001-07-17
影响因子：
11.1
作者：
Chmura, AJ;Orton, MS;Meares, CF
通讯作者：
Meares, CF
影响因子：
15
作者：
Nonaka, Hiroshi;Tsukiji, Shinya;Hamachi, Itaru
通讯作者：
Hamachi, Itaru
影响因子：
3.2
作者：
Krusemark, Casey J.;Belshaw, Peter J.
通讯作者：
Belshaw, Peter J.