Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM.
Identifying and quantifying two ligand-binding sites while imaging native human membrane receptors by AFM.
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DOI：
10.1038/ncomms9857
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发表时间：
2015-11-12
影响因子：
16.6
通讯作者：
Müller DJ
中科院分区：
文献类型：
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作者：
Pfreundschuh M;Alsteens D;Wieneke R;Zhang C;Coughlin SR;Tampé R;Kobilka BK;Müller DJ
生命科学中的当前挑战是对细胞膜受体进行成像，同时表征它们与各种配体的特异性相互作用。解决这个问题一直受到缺乏合适的纳米方法的阻碍。在这里，我们解决这一挑战，并引入多功能高分辨率原子力显微镜（AFM）的图像人类蛋白酶激活受体（PAR 1）在功能上重要的脂质膜，并同时定位和量化其结合到两个不同的配体。因此，我们引入了表面化学双功能化的原子力显微镜提示与天然受体激活肽和三-N-次氮基三乙酸（三-NTA）组结合到His 10标签工程PAR 1。我们进一步介绍的方法来辨别这两种配体的结合不同的受体网站，同时成像天然PAR 1。表面化学和纳米方法适用于一系列生物系统在体外和体内，并同时检测和定位多个配体结合位点在单一受体分辨率。 膜蛋白的功能分析将受益于成像与配体表征的结合。在这里，Pfreundschuh等人使用专门的原子力显微镜尖端同时对两种配体的结合进行成像和定量。
A current challenge in life sciences is to image cell membrane receptors while characterizing their specific interactions with various ligands. Addressing this issue has been hampered by the lack of suitable nanoscopic methods. Here we address this challenge and introduce multifunctional high-resolution atomic force microscopy (AFM) to image human protease-activated receptors (PAR1) in the functionally important lipid membrane and to simultaneously localize and quantify their binding to two different ligands. Therefore, we introduce the surface chemistry to bifunctionalize AFM tips with the native receptor-activating peptide and a tris-N-nitrilotriacetic acid (tris-NTA) group binding to a His10-tag engineered to PAR1. We further introduce ways to discern between the binding of both ligands to different receptor sites while imaging native PAR1s. Surface chemistry and nanoscopic method are applicable to a range of biological systems in vitro and in vivo and to concurrently detect and localize multiple ligand-binding sites at single receptor resolution. Functional analysis of membrane proteins would benefit from the combination of imaging with ligand characterisation. Here, Pfreundschuh et al. use specialised atomic force microscopy tips to image and quantify the binding of two ligands at the same time.