From Nanodiscs to Isotropic Bicelles: A Procedure for Solution Nuclear Magnetic Resonance Studies of Detergent-Sensitive Integral Membrane Proteins
From Nanodiscs to Isotropic Bicelles: A Procedure for Solution Nuclear Magnetic Resonance Studies of Detergent-Sensitive Integral Membrane Proteins
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DOI：
10.1016/j.str.2016.07.017
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发表时间：
2016-10-04
期刊：
影响因子：
5.7
通讯作者：
Doetsch, Volker
中科院分区：
文献类型：
--
作者：
Laguerre, Aisha;Loehr, Frank;Doetsch, Volker
纳米盘和各向同性的双胞是在膜蛋白的溶液核磁共振（NMR）光谱领域中有前途的膜模拟物。尽管IMP的溶液NMR研究面临各种挑战，但我们将这些环境中溶液NMR结构的缺乏归因于各种IMP在标准纳米盘和bicelle制备过程中无法承受洗涤剂处理。在这里，我们提出了一种策略，创建小的各向同性bicelles从IMP co-conflicently嵌入在大nanodisces使用无细胞表达。我们的研究结果表明，在保留脂质IMP接触的同时，NMR光谱质量有明显的提高。我们验证了洗涤剂敏感的LspA，这最终使我们能够进行高质量的三重共振NMR实验的结构研究的方法。我们的策略生产bicelles从nanodisks全面避免洗涤剂在表达和制备过程中，是适合的溶液NMR光谱的脂质-IMP复合物。
Nanodiscs and isotropic bicelles are promising membrane mimetics in the field of solution nuclear magnetic resonance (NMR) spectroscopy of integral membrane proteins (IMPs). Despite varied challenges to solution NMR studies of IMPs, we attribute the paucity of solution NMR structures in these environments to the inability of diverse IMPs to withstand detergent treatment during standard nanodisc and bicelle preparations. Here, we present a strategy that creates small isotropic bicelles from IMPs co-translationally embedded in large nanodiscs using cell-free expression. Our results demonstrate appreciable gains in NMR spectral quality while preserving lipid-IMP contacts. We validate the approach on the detergent-sensitive LspA, which finally allowed us to perform high-quality triple-resonance NMR experiments for structural studies. Our strategy of producing bicelles from nanodiscs comprehensively avoids detergent during expression and preparation and is suitable for solution NMR spectroscopy of lipid-IMP complexes.