Light-Up "Channel Dyes" for Haloalkane-Based Protein Labeling in Vitro and in Bacterial Cells.
Light-Up "Channel Dyes" for Haloalkane-Based Protein Labeling in Vitro and in Bacterial Cells.
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在体外和细菌细胞中对基于卤代烷基的蛋白质标记的点亮“通道染料”。
DOI：
10.1021/acs.bioconjchem.6b00613
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发表时间：
2016-12-21
影响因子：
4.7
通讯作者：
Kool ET
中科院分区：
文献类型：
--
作者：
Clark SA;Singh V;Vega Mendoza D;Margolin W;Kool ET
我们描述了一种新的分子策略，用于在遗传编码的HaloTag蛋白结构域的荧光标记中产生强亮信号。我们设计了一组具有扭曲内部电荷转移（TICT）结构的卤代烷衍生染料，其可能足够窄，可以部分适应酶的卤代烷结合通道。对一系列短链长度的测试揭示了许多活性染料，其中7个碳产生了最佳的发光信号。二甲氨基二唑氯庚基染料（1d）与蛋白质反应后荧光发射增强27倍（λex = 535 nm; Em(max) = 616 nm）。具有标准12原子键的对照化合物表现出较低的信号传导效率，与我们的通道结合假设一致。为了进一步向红光发射，我们还制备了一种氯庚基萘染料；化合物2在653 nm处发光，与HaloTag结构域反应后荧光增强。这两种染料（1d, 2）在细菌中蛋白定位的无水洗成像中成功测试，使用HaloTag融合大肠杆菌（E. coli）中丝状温度敏感突变体Z （FtsZ）蛋白。新的染料偶联物是廉价和容易合成的酶底物，具有低背景和大斯托克斯位移，为HaloTag酶提供了比已知荧光底物更大的优势。
We describe a novel molecular strategy for engendering a strong light-up signal in fluorescence tagging of the genetically encoded HaloTag protein domain. We designed a set of haloalkane-derivatized dyes having twisted internal charge transfer (TICT) structures potentially narrow enough to partially fit into the enzyme’s haloalkane-binding channel. Testing a range of short chain lengths revealed a number of active dyes, with seven carbons yielding optimum light-up signal. The dimethylaminostilbazolium chloroheptyl dye (1d) yields a 27-fold fluorescence emission enhancement (λex = 535 nm; Em(max) = 616 nm) upon reaction with the protein. The control compound with standard 12-atom linkage shows less efficient signaling, consistent with our channel-binding hypothesis. For emission further to the red, we also prepared a chloroheptyl naphthalene-based dye; compound 2 emits at 653 nm with strong fluorescence enhancement upon reaction with the HaloTag domain. The two dyes (1d, 2) were successfully tested in wash-free imaging of protein localization in bacteria, using a HaloTag fusion of the filamenting temperature-sensitive mutant Z (FtsZ) protein in Escherichia coli (E. coli). The new dye conjugates are inexpensive and easily synthesized enzyme substrates with low background and large Stokes shifts, offering substantial benefits over known fluorescent substrates for the HaloTag enzyme.
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DOI：
10.1038/nrmicro.2016.26
发表时间：
2016-04
期刊：
Nature reviews. Microbiology
影响因子：
--
作者：
Haeusser DP;Margolin W
通讯作者：
Margolin W
影响因子：
4.7
作者：
Naganbabu M;Perkins LA;Wang Y;Kurish J;Schmidt BF;Bruchez MP
通讯作者：
Bruchez MP
影响因子：
4
作者：
Liu, Tao-Kai;Hsieh, Pei-Ying;Tan, Kui-Thong
通讯作者：
Tan, Kui-Thong
DOI：
10.1073/pnas.93.23.12998
发表时间：
1996-11-12
影响因子：
11.1
作者：
Ma, XL;Ehrhardt, DW;Margolin, W
通讯作者：
Margolin, W
影响因子：
64.5
作者：
WARD, JE;LUTKENHAUS, J
通讯作者：
LUTKENHAUS, J