GPCR stabilization using the bicelle-like architecture of mixed sterol-detergent micelles.
GPCR stabilization using the bicelle-like architecture of mixed sterol-detergent micelles.
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DOI：
10.1016/j.ymeth.2011.10.011
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发表时间：
2011-12
期刊：
影响因子：
4.8
通讯作者：
Stevens, Raymond C.
中科院分区：
文献类型：
--
作者：
Thompson, Aaron A.;Liu, Jeffrey J.;Chun, Eugene;Wacker, Daniel;Wu, Huixian;Cherezov, Vadim;Stevens, Raymond C.
纯化的膜蛋白的生物物理表征通常需要从天然脂质膜环境中进行去污剂介导的提取。在人类G蛋白偶联受体（GPCR）的情况下，这一过程已经复杂化，其构象异质性和普遍缺乏了解的组成和相互作用内的不同的人类细胞膜环境。几个成功的GPCR结构测定的努力表明，胆固醇类似物的添加往往是维持蛋白质稳定性的关键。我们已经鉴定出甾醇可以大幅增加阿片类GPCR家族成员NOP受体（ORL-1）在混合胶束环境中的稳定性。使用动态光散射和小角度X-射线散射，我们已经确定，最热稳定甾醇，胆固醇hemmisubicate，诱导形成一个bicelle样胶束结构时，与十二烷基麦芽糖苷洗涤剂混合。连同诱变研究和最近的GPCR结构，我们的研究结果提供的迹象表明，稳定是通过结合特定的甾醇GPCR和胶束形态的调制。
The biophysical characterization of purified membrane proteins typically requires detergent mediated extraction from native lipid membrane environments. In the case of human G protein-coupled receptors (GPCRs), this process has been complicated by their conformational heterogeneity and the general lack of understanding the composition and interactions within the diverse human cellular membrane environment. Several successful GPCR structure determination efforts have shown that the addition of cholesterol analogs is often critical for maintaining protein stability. We have identified sterols that substantially increase the stability of the NOP receptor (ORL-1), a member of the opioid GPCR family, in a mixed micelle environment. Using dynamic light scattering and small-angle X-ray scattering, we have determined that the most thermal stabilizing sterol, cholesteryl hemmisuccinate, induces the formation of a bicelle-like micelle architecture when mixed with dodecyl maltoside detergent. Together with mutagenesis studies and recent GPCR structures, our results provide indications that stabilization is attained through a combination of specific sterol binding to GPCRs and modulation of micelle morphology.
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