ACS Nano
·
2024
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IF 15.8
Peng Xu,Simei Zhong,Yunfei Wei,Xinxin Duan,Meng Zhang,Wei Shen,Yan Ma,Yu-Hui Zhang
Super-resolution fluorescence microscopy has emerged as a powerful tool for studying endoplasmic reticulum (ER) dynamics in living cells. However, the lack of high-brightness, high-photostability, and stable labeling probes makes long-term super-resolution imaging of the ER still challenging. Herein, we reported a surface-functionalized Halo-tag gold nanofluorescent probe (GNP-Atto565-fR8-CA) that exhibits excellent brightness, photostability, and biocompatibility. GNP-Atto565-fR8-CA can simultaneously load multiple Atto565 dye molecules, significantly improving its brightness. Modifying the cell-penetrating peptide fR8 enables GNP-Atto565-fR8-CA to be efficiently delivered into the cytoplasm, overcoming the challenge of their easy entrapment in vesicles. Fluorescent labeling of ER proteins via Halo tags enables high specificity and stable labeling of GNP-Atto565-fR8-CA to the ER. The SIM super-resolution imaging results showed that GNP-Atto565-fR8-CA can track and observe the long-term dynamic process of the ER, and can also be used for long-term super-resolution imaging of the dynamic interactions between the ER and other organelles. This work offers a practical tool to study live-cell ER ultrastructure and dynamics.
研究领域:
细胞生物学
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分子影像学
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生物化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF CHROMATOGRAPHY A
·
2021
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IF 4.759
Xiaoying Fu,Linkang Li,Xin Wen,Ru Xu,Yan Xue,Haiyue Zuo,Qi Liang,Gangjun Feng,Jing Wang,Xinfeng Zhao
In previous work, we have established a one-step method to immobilize halo-tagged proteins onto microspheres through the covalent bond formed between the halo-tag and the halide linkers on the support surface. We observe extremely tailed peaks of most of drugs on the immobilized proteins, which is reasoned by the nonspecific interaction between the linkers and the drugs. To prove this, the current work designed five different halide linkers for the immobilization of beta 2 -adrenoceptor (β 2 -AR). We applied the immobilized receptor to systematically realize the effects of these halide linkers on drug-receptor interaction by analyzing peak profiles of five drugs. The retention times and the half-widths of the drugs appeared to be negatively correlated to the atom numbers of the linkers in the range of 6–13 atoms. Subsequent increase of linker atoms resulted in reduced retention times and wider peaks of the drugs. Applying identical linker length, we observed clear reduced retention times and half-widths of the five drugs than the linker in the absence of oxygen atom. Such improvement was dominated by the number of oxygen atoms. These indicated that linker S-4 (2-(2-(2-(2-chloroethoxy) ethoxy) ethoxy) acetic acid) was optimal to eliminate the unwanted non-specific interactions. In comparison with the columns prepared by linker S-1 (6-chlorocaproic acid) and histidine tagged β 2 -AR, the drugs on the linker S-4 column gave greater dissociation rate constants (e.g. 60.3±0.3 s -1 for salbutamol), which is closer to the data in literatures. Taking together, we concluded that optimization of the linker structure plays particular role in reducing the non-specific interaction between the immobilized protein and the drugs, thereby making the determination of drug-protein interaction more reliable.
研究领域:
生物化学
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药物化学
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分析化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF CHROMATOGRAPHY A
·
2021
·
IF 4.759
Xue Zhao,Xiaoying Fu,Xinyi Yuan,Aerduosi Shayiranbieke,Ru Xu,Fang Cao,Jianping Ren,Qi Liang,Xinfeng Zhao
The pursuit of new ligands binding to muscarinic-3 acetylcholine receptor (M 3 R) is viewed as challenging due to the lack of screening methods with high efficiency. To address such challenges, this work developed and characterized an approach to the rapid discovery of M 3 R ligands using the immobilized receptor as the chromatographic stationary phase. We fused haloalkane dehalogenase (Halo) as a tag at the C-terminus of M 3 R. The fusion M 3 R was immobilized on 6-chlorocaproic acid-activated ammino-microspheres by the specific covalent reaction between the Halo-tag and the linker. Comprehensive characterizations of the immobilized M 3 R were performed by scanning electron microscope, X-ray photoelectron spectroscopy, and the investigation on the binding of three specific ligands to the receptor. The feasibility of the immobilized M 3 R in complex matrices was tested by screening the bioactive compounds in Zhisou oral liquid, assessing the interaction between the screened compounds and the receptor using zonal elution, and evaluating the in vivo activity of the targeted compounds. The results evidenced that the immobilized M 3 R has high specificity, good stability, and the capacity to separate M 3 R ligands from complex matrices. These allowed us to identify naringin, hesperidin, liquiritigenin, platycodin D, and glycyrrhizic acid as the potential ligands of M 3 R. The association constants of the five compounds to M 3 R were 4.44 × 10 4 , 1.11 × 10 4 , 7.20 × 10 4 , 4.15 × 10 4 , and 3.36 × 10 4 M −1 . The synergistic application of the five compounds exhibited an equivalent expectorant activity to the original formula. We reasoned that the current method is possible to provide a highly efficient strategy for the discovery of receptor ligands.
研究领域:
药理学
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分析化学
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药物发现
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）