The Cation-π Interaction Enables a Halo-Tag Fluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-Free Protein Quantification.
The Cation-π Interaction Enables a Halo-Tag Fluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-Free Protein Quantification.
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DOI：
10.1021/acs.biochem.7b00056
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发表时间：
2017-03-21
期刊：
影响因子：
2.9
通讯作者：
Zhang X
中科院分区：
文献类型：
--
作者：
Liu Y;Miao K;Dunham NP;Liu H;Fares M;Boal AK;Li X;Zhang X
用于Halo标签的荧光探针的设计是非常可取的，但也具有挑战性。以前的工作是通过在Halo标签和探针之间的共价连接上控制螺内酯的化学开关或通过在Halo标签的底物结合隧道中掺入通道染料来实现这一目标的。在这项工作中，我们开发了一类新型的卤素标记荧光探针，它们是从溶剂变色荧光团衍生而来的。最优的探针含有苯并噻二唑支架，与Halo标签反应时，荧光增强了1000倍。结构、计算和生物化学研究表明，色氨酸残基的苯环在激发态与苯并噻二唑荧光团的二甲氨基给电子基发生阳离子−π相互作用。我们进一步用非正则氟化色氨酸证明了阳离子−π相互作用直接影响了苯并噻二唑荧光团的生氟性。从机理上讲，这种相互作用可能通过促进激发态电荷分离和抑制二甲氨基的扭曲运动来促进生氟性，从而导致生氟性的增强。最后，我们展示了该探针在活体细胞中直接成像Halo标记蛋白质的实用性。此外，该探针的荧光性质使得能够对哺乳动物细胞中表达的融合蛋白进行无胶量化，这是以前的非荧光Halo-Tag探针无法实现的应用。这项工作揭示的独特机制表明，引入激发态阳离子−π相互作用可能是提高荧光团和荧光传感器光学性能的一种可行策略。
The design of fluorogenic probes for a Halo tag is highly desirable but challenging. Previous work achieved this goal by controlling the chemical switch of spirolactones upon the covalent conjugation between the Halo tag and probes or by incorporating a “channel dye” into the substrate binding tunnel of the Halo tag. In this work, we have developed a novel class of Halo-tag fluorogenic probes that are derived from solvatochromic fluorophores. The optimal probe, harboring a benzothiadiazole scaffold, exhibits a 1000-fold fluorescence enhancement upon reaction with the Halo tag. Structural, computational, and biochemical studies reveal that the benzene ring of a tryptophan residue engages in a cation−π interaction with the dimethylamino electron-donating group of the benzothiadiazole fluorophore in its excited state. We further demonstrate using noncanonical fluorinated tryptophan that the cation−π interaction directly contributes to the fluorogenicity of the benzothiadiazole fluorophore. Mechanistically, this interaction could contribute to the fluorogenicity by promoting the excited-state charge separation and inhibiting the twisting motion of the dimethylamino group, both leading to an enhanced fluorogenicity. Finally, we demonstrate the utility of the probe in no-wash direct imaging of Halo-tagged proteins in live cells. In addition, the fluorogenic nature of the probe enables a gel-free quantification of fusion proteins expressed in mammalian cells, an application that was not possible with previously nonfluorogenic Halo-tag probes. The unique mechanism revealed by this work suggests that incorporation of an excited-state cation−π interaction could be a feasible strategy for enhancing the optical performance of fluorophores and fluorogenic sensors.