Aptamer-assisted two-point immobilized agonist-bound angiotensin II type 1 receptor for a second-site modulator discovery.
Aptamer-assisted two-point immobilized agonist-bound angiotensin II type 1 receptor for a second-site modulator discovery.
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DOI：
10.1016/j.isci.2022.105361
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发表时间：
2022-11-18
期刊：
影响因子：
5.8
通讯作者：
Zhao, Xinfeng
中科院分区：
文献类型：
--
作者：
Liu, Jiajun;Li, Ting;Wang, Ge;Chen, Jiahuan;Yao, Qingqing;Li, Qian;Zhao, Xinfeng
固定化蛋白质的方法面临着如何捕获处于完整功能状态的蛋白质的挑战。在这里，我们提出了一种两点的、高特异性的方法，将构象特异性的血管紧张素II 1型受体(AT1R)固定在氨基功能化的聚苯乙烯微球上。我们通过具有微摩尔亲和力的柱基SELEX方法鉴定了AT1R的一个选择性DNA适配子。利用两条单链DNA链将AT1R适配子和血管紧张素II 3-8肽引入微球表面，形成两个表面定位的位点。与单点固定化AT1R相比，两点固定化AT1R具有更高的配体结合活性和稳定性。从中草药提取物中筛选出人参皂苷Rg1和迷迭香酸，证实它们分别通过受体的变构和邻位与AT1R结合。这为功能蛋白固定化提供了一种普遍适用的方法，具有更高的构象稳定性、配体结合活性和筛选效率。提出了一种构象选择性适体筛选的在线SELEX方法，介绍了用于功能蛋白固定化的适体和多肽，利用亲和层析同时筛选拮抗剂和变构配体从中草药提取物中首次报道了一种潜在的变构调节剂生物化学；生物分子；结构生物学
Methods of immobilized proteins are challenged by the way how to capture the proteins in their intact functional states. Here we present a two-point, high-specific method for the immobilization of conformationally specific angiotensin II type 1 receptor (AT1R) on amino-functionalized polystyrene microspheres. We identified a selective DNA aptamer of AT1R by a column-based SELEX approach with micromolar affinity. Two single-stranded DNA strands were utilized to introduce the AT1R aptamer and angiotensin II 3-8 peptide to the microsphere surface, resulting in the two surface-positioned sites. The two-point immobilized AT1R exhibited enhanced ligand-binding activity and stability in comparison with that prepared by a one-positioned site. Ginsenoside Rg1 and rosmarinic acid were screened from the herbal extract and proved to bind with AT1R through the allosteric and orthosteric sites of the receptor, respectively. These provide a generally applicable approach for functional protein immobilization with enhanced conformation stability, ligand binding activity, and screening efficiency. An online SELEX approach was proposed for conformation selective aptamer screening Aptamer and peptide were introduced for functional protein immobilization Simultaneous screening of antagonists and allosteric ligands by affinity chromatography A potential allosteric modulator was first reported from the herbal extract Biochemistry; Biomolecules; Structural biology
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影响因子：
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作者：
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影响因子：
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DOI：
10.1073/pnas.2019126118
发表时间：
2021-08-17
影响因子：
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通讯作者：
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