Overexpression and functional characterization of the extracellular domain of the human alpha1 glycine receptor.
Overexpression and functional characterization of the extracellular domain of the human alpha1 glycine receptor.
复制标题
人 α1 甘氨酸受体胞外域的过表达和功能表征。
DOI：
10.1021/bi800659x
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发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
Cascio,Michael
中科院分区：
文献类型：
--
作者：
Liu,Zhenyu;Ramanoudjame,Gomathi;Liu,Deqian;Fox,RobertO;Jayaraman,Vasanthi;Kurnikova,Maria;Cascio,Michael
使用杆状病毒表达系统过表达人α1甘氨酸受体（GlyR）配体结合胞外结构域（ECD）的一种新截短形式（残基1 - 214，具有随机C末端尾），其中乙酰胆碱结合蛋白（AChBP）相应序列的氨基酸取代了两个相对疏水的近膜环。的突变体GlyR ECD，命名为GlyBP，存在于细胞裂解后的可溶性和膜相关的馏分，虽然只有后者似乎是在一个天然的构象能够结合士的宁，GlyR特异性拮抗剂。溶解膜相关的GlyBP，亲和纯化去污剂/脂质/蛋白质胶束。在去污剂去除后，GlyBP可以以水性或囊泡形式分离。结合试验和光谱研究，使用圆二色性和FRET是一致的，这两种形式采用等效的天然样构象。因此，GlyBP可以分离为可溶性或膜相关组件，其充当GlyR的ECD的结构和功能同源物。
A novel truncated form (residues 1−214, with a randomized C-terminal tail) of the ligand-binding extracellular domain (ECD) of the human α1 glycine receptor (GlyR), with amino acids from the corresponding sequence of an acetylcholine binding protein (AChBP) substituted for two relatively hydrophobic membrane-proximal loops, was overexpressed using a baculovirus expression system. The mutant GlyR ECD, named GlyBP, was present in both soluble and membrane-associated fractions after cell lysis, though only the latter appeared to be in a native-like conformation capable of binding strychnine, a GlyR specific antagonist. The membrane-associated GlyBP was solubilized, and detergent/lipid/protein micelles were affinity purified. After detergent removal, GlyBP may be isolated in either aqueous or vesicular form. Binding assays and spectroscopic studies using circular dichroism and FRET are consistent with both forms adopting equivalent native-like conformations. Thus, GlyBP may be isolated as a soluble or membrane-associated assembly that serves as a structural and functional homologue of the ECD of GlyR.