5uxz
X-ray crystal structure of Halotag bound to the P9 benzothiadiazole fluorogenic ligand
Structural highlights
FunctionDHAA_RHORH Catalyzes hydrolytic cleavage of carbon-halogen bonds in halogenated aliphatic compounds, leading to the formation of the corresponding primary alcohols, halide ions and protons. Expresses halogenase activity against 1-chloroalkanes of chain length C3 to C10, and also shows a very weak activity with 1,2-dichloroethane. Publication Abstract from PubMedDesign 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-pi 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-pi 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-pi interaction could be a feasible strategy to enhance optical performance of fluorophores and fluorogenic sensors. The Cation-pi Interaction Enables a Halo-Tag Fluorogenic Probe for Fast No-Wash Live Cell Imaging and Gel-Free Protein Quantification.,Liu Y, Miao K, Dunham NP, Liu H, Fares M, Boal AK, Li X, Zhang X Biochemistry. 2017 Feb 21. doi: 10.1021/acs.biochem.7b00056. PMID:28221782[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
| ||||||||||||||||||||
