Analyzing native membrane protein assembly in nanodiscs by combined non-covalent mass spectrometry and synthetic biology
Analyzing native membrane protein assembly in nanodiscs by combined non-covalent mass spectrometry and synthetic biology
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DOI：
10.7554/elife.20954
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发表时间：
2017-01-09
期刊：
影响因子：
7.7
通讯作者：
Morgner, Nini
中科院分区：
文献类型：
--
作者：
Henrich, Erik;Peetz, Oliver;Morgner, Nini
膜蛋白经常在其天然脂环境中组装成更高阶的同源或异源低聚物。这种络合物的形成可以调节它们的折叠、活性以及底物的选择性。因此，避免膜崩解、转移到人工环境或化学修饰等关键步骤的非破坏性方法对于分析天然膜蛋白组装的分子机制是必不可少的。无细胞合成生物学、纳米盘技术和非共价质谱技术的结合为膜蛋白在限定膜内齐聚的分析提供了极好的协同作用。我们通过对各种膜蛋白的寡聚态表征来举例说明我们的策略，这些膜蛋白包括离子通道、转运体和组装成六聚体复合体的膜集成酶。我们进一步指出，mray转位酶的脂质依赖二聚体的形成与酶活性有关。膜蛋白/纳米盘样品的免洗涤剂合成和LILBID质谱学分析为天然环境中膜蛋白的分析提供了一个通用的平台。
Membrane proteins frequently assemble into higher order homo- or hetero-oligomers within their natural lipid environment. This complex formation can modulate their folding, activity as well as substrate selectivity. Non-disruptive methods avoiding critical steps, such as membrane disintegration, transfer into artificial environments or chemical modifications are therefore essential to analyze molecular mechanisms of native membrane protein assemblies. The combination of cell-free synthetic biology, nanodisc-technology and non-covalent mass spectrometry provides excellent synergies for the analysis of membrane protein oligomerization within defined membranes. We exemplify our strategy by oligomeric state characterization of various membrane proteins including ion channels, transporters and membrane-integrated enzymes assembling up to hexameric complexes. We further indicate a lipid-dependent dimer formation of MraY translocase correlating with the enzymatic activity. The detergent-free synthesis of membrane protein/nanodisc samples and the analysis by LILBID mass spectrometry provide a versatile platform for the analysis of membrane proteins in a native environment.