A transmembrane helix-bundle from G-protein coupled receptor CB2: biosynthesis, purification, and NMR characterization.
A transmembrane helix-bundle from G-protein coupled receptor CB2: biosynthesis, purification, and NMR characterization.
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G 蛋白偶联受体 CB2 的跨膜螺旋束：生物合成、纯化和 NMR 表征。
DOI：
10.1002/bip.20526
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Xie,Xiang-Qun
中科院分区：
文献类型：
--
作者：
Zheng,HaiAn;Zhao,Ju;Sheng,Wanyun;Xie,Xiang-Qun
大麻素受体亚型2（CB 2）是G蛋白偶联受体（GPCR）超家族的成员。由于这种受体的结构和功能之间的关系仍然知之甚少，本研究进行了表征的一段，包括第一和第二跨膜螺旋（TM 1和TM 2）域的CB 2的结构。为了实现这一点，表达、纯化并通过NMR表征来自该区域的跨膜双螺旋束。使用融合蛋白过表达策略生物合成毫克该受体的疏水片段，并通过亲和色谱结合反相HPLC纯化。实施化学和酶切方法以去除融合标签。通过HPLC、质谱和圆二色性（CD）分析和确认所得重组蛋白样品。溶液和DPC胶束制剂中HPLC纯化蛋白的CD分析表明，在两种条件下均存在主要的α螺旋结构。13 C/15 N双标记的蛋白质CB 2（27-101）通过NMR光谱进一步验证和分析。完成了80%以上的残基的顺序分配。15 N HSQC NMR结果显示酰胺氮-质子相关性的明确化学位移分散，表明纯双标记多肽分子。结果表明，该方法能够从GPCR产生足够用于NMR和其他生物物理研究的量和纯度的跨膜螺旋束。因此，GPCR跨膜螺旋束的生物合成代表了从重组“构件”获得和组装NMR结构的令人满意的替代策略。© 2006 Wiley Periodicals，Inc. Biopolymers 83：46-61，2006这篇文章最初作为公认的预印本在线发表。“在线发布”日期对应于预印本。您可以通过向Biopolymers编辑部发送电子邮件（biopolymers@wiley.com）索取预印本的副本
The cannabinoid receptor subtype 2 (CB2) is a member of the G‐protein coupled receptor (GPCR) superfamily. As the relationship between structure and function for this receptor remains poorly understood, the present study was undertaken to characterize the structure of a segment including the first and second transmembrane helix (TM1 and TM2) domains of CB2. To accomplish this, a transmembrane double‐helix bundle from this region was expressed, purified, and characterized by NMR. Milligrams of this hydrophobic fragment of the receptor were biosynthesized using a fusion protein overexpression strategy and purified by affinity chromatography combined with reverse phase HPLC. Chemical and enzymatic cleavage methods were implemented to remove the fusion tag. The resultant recombinant protein samples were analyzed and confirmed by HPLC, mass spectrometry, and circular dichroism (CD). The CD analyses of HPLC‐purified protein in solution and in DPC micelle preparations suggested predominant α‐helical structures under both conditions. The13C/15N double‐labeled protein CB2(27–101) was further verified and analyzed by NMR spectroscopy. Sequential assignment was accomplished for more than 80% of residues. The15N HSQC NMR results show a clear chemical shift dispersion of the amide nitrogen–proton correlation indicative of a pure double‐labeled polypeptide molecule. The results suggest that this method is capable of generating transmembrane helical bundles from GPCRs in quantity and purity sufficient for NMR and other biophysical studies. Therefore, the biosynthesis of GPCR transmembrane helix bundles represents a satisfactory alternative strategy to obtain and assemble NMR structures from recombinant “building blocks.” © 2006 Wiley Periodicals, Inc. Biopolymers 83: 46–61, 2006This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com