A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies.
A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies.
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一种诱变和筛选策略，以生成最佳的恒温膜蛋白进行结构研究。
DOI：
10.1038/nprot.2016.088
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发表时间：
2016-08
期刊：
影响因子：
14.8
通讯作者：
Tate CG
中科院分区：
文献类型：
--
作者：
Magnani F;Serrano-Vega MJ;Shibata Y;Abdul-Hussein S;Lebon G;Miller-Gallacher J;Singhal A;Strege A;Thomas JA;Tate CG
完整膜蛋白在洗涤剂溶液中的热稳定性是决定获得适合于结构测定的良好衍射晶体的可能性的关键参数。然而，许多哺乳动物膜蛋白太不稳定而不能结晶。我们开发了一种热稳定化策略的基础上系统诱变耦合到放射性配体结合热稳定性测定，可应用于受体，离子通道和转运蛋白。需要大约6-12个月的时间来热稳定含有300个氨基酸残基的G蛋白偶联受体（GPCR）。由此产生的热稳定膜蛋白更容易结晶并产生高质量的结构。这种方法促进了应用于GPCR的基于结构的药物设计，因为可以确定与低亲和力配体结合的热稳定受体的多种结构。如何开发膜蛋白的热稳定性测定和如何联合收割机突变，使最佳稳定的突变体适合结构研究的协议和建议。
The thermostability of an integral membrane protein in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals suitable for structure determination. However, many mammalian membrane proteins are too unstable for crystallisation. We developed a thermostabilisation strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes approximately 6-12 months to thermostabilise a G protein-coupled receptor (GPCR) containing 300 amino acid residues. The resulting thermostabilised membrane proteins are more easily crystallised and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs, because it is possible to determine multiple structures of the thermostabilised receptors bound to low affinity ligands. Protocols and advice are given on how to develop thermostability assays for membrane proteins and how to combine mutations to make an optimally stable mutant suitable for structural studies.