G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent.
G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent.
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DOI：
10.1042/bsr20140171
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发表时间：
2015-04-16
影响因子：
4
通讯作者：
Wheatley M
中科院分区：
文献类型：
--
作者：
Jamshad M;Charlton J;Lin YP;Routledge SJ;Bawa Z;Knowles TJ;Overduin M;Dekker N;Dafforn TR;Bill RM;Poyner DR;Wheatley M
G蛋白偶联受体（GPCR）构成最大类的膜蛋白，并且是主要的药物靶标。研究GPCR结构/功能特征的一个严重障碍是需要从质膜中的天然环境中提取受体，再加上GPCR在其溶解所需的去污剂中的固有不稳定性。在本研究中，我们报告的第一个溶解和纯化的功能GPCR [人腺苷A2 A受体（A2 AR）]，在完全没有洗涤剂在任何阶段，通过利用自发封装苯乙烯马来酸（SMA）共聚物直接从膜到纳米级SMA脂质颗粒（SMALP）。此外，与去污剂[DDM（正十二烷基-β-D-吡喃麦芽糖苷）]-溶解的A2 AR对照相比，由酵母（巴斯德毕赤酵母）或哺乳动物细胞产生的A2 AR-SMALP表现出增加的热稳定性（105 °C）。A2 AR-SMALP在4°C下长时间储存时也是稳定的，并且耐多次冻融循环，与洗涤剂溶解的受体形成鲜明对比。这些性质确立了在基于受体的药物发现测定中使用GPCR-SMALP的潜力。此外，与由支架蛋白稳定的纳米盘相比，SMA聚合物的非蛋白质性质允许嵌入的受体的清晰的生物物理表征。因此，CD光谱用于将二级结构的变化与配体结合（[3 H] ZM 241385）能力的丧失相关联。GPCR的SMALP增溶，保留环状脂质环境，将使广泛的治疗靶标能够以天然样状态制备，以帮助药物发现和理解GPCR分子机制。众所周知，将细胞表面受体暴露于去污剂是有害的。我们已经使用聚合物提取受体和周围的脂质作为纳米颗粒，提供了一种新的解决方案，与纯化和基于受体的药物发现测定兼容。
G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A2A receptor (A2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A2AR–SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A2AR controls. The A2AR–SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR–SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([3H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms. It is universally acknowledged that exposing cell-surface receptors to detergent is detrimental. We have used a polymer to extract the receptor and surrounding lipid as a nanoparticle that provides a novel solution compatible with purification and receptor-based drug discovery assays.