Zheng HaiAn, Zhao Ju, Sheng Wanyun, Xie Xiang-Qun
Department of Pharmaceutical and Pharmacological Sciences, College of Pharmacy, University of Houston, Houston, TX, 77204-5037, USA.
Biopolymers. 2006 Sep;83(1):46-61. doi: 10.1002/bip.20526.
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 alpha-helical structures under both conditions. The 13C/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. The 15N 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."
大麻素受体亚型2（CB2）是G蛋白偶联受体（GPCR）超家族的成员。由于对该受体的结构与功能之间的关系仍了解甚少，因此开展了本研究以表征包含CB2的第一和第二跨膜螺旋（TM1和TM2）结构域的片段的结构。为实现这一目标，表达、纯化了来自该区域的跨膜双螺旋束，并通过核磁共振（NMR）对其进行了表征。利用融合蛋白过表达策略生物合成了毫克级的该受体疏水片段，并通过亲和色谱结合反相高效液相色谱（HPLC）进行了纯化。采用化学和酶切方法去除融合标签。通过HPLC、质谱和圆二色性（CD）对所得重组蛋白样品进行了分析和确认。对溶液中和DPC胶束制剂中的HPLC纯化蛋白进行的CD分析表明，在这两种条件下主要为α-螺旋结构。对13C/15N双标记蛋白CB2(27 - 101)进一步通过核磁共振波谱进行了验证和分析。完成了80%以上残基的序列归属。15N HSQC NMR结果显示酰胺氮-质子相关性的明显化学位移分散，表明是一个纯的双标记多肽分子。结果表明，该方法能够大量生成纯度足以用于NMR和其他生物物理研究的GPCR跨膜螺旋束。因此，GPCR跨膜螺旋束的生物合成是一种从重组“构建模块”获得并组装NMR结构的令人满意的替代策略。
