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The Cation-π Interaction Enables a Halo-Tag Fluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-Free Protein Quantification.

著者 Liu Y , Miao K , Dunham NP , Liu H , Fares M , Boal AK , Li X , Zhang X
Biochemistry.2017 Feb 21 ; ():.
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Design of fluorogenic probes for Halo-tag is highly desirable but challenging. Previous work achieved this goal by controlling the chemical switch of spirolactones upon the covalent conju-gation between Halo-tag and probes or by incorporating a 'channel dye' into the substrate bind-ing tunnel of 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 ben-zothiadiazole scaffold, exhibits a 1000-fold fluorescence enhancement upon reaction with Halo-tag. Structural, computational and biochemical studies reveal that the benzene ring of a trypto-phan 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 non-canonical fluorinated tryptophan that the cation-π interaction directly contributes to fluorogenici-ty of the benzothiadiazole fluorophore. Mechanistically, this interaction could contribute to the fluorogenicity by promoting the excited-state charge separation and inhibiting the twisting mo-tion of the dimethyl-amino 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 non-fluorogenic Halo-tag probes. The unique mechanism revealed by this work suggests that incor-poration of an excited-state cation-π interaction could be a feasible strategy to enhance optical performance of fluorophores and fluorogenic sensors.
PMID: 28221782 [PubMed - as supplied by publisher]
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