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Proton-Detected Solid-State NMR of the Cell-Free Synthesized α-Helical Transmembrane Protein NS4B from Hepatitis C Virus.
ChemBioChem ( IF 2.6 ) Pub Date : 2020-02-20 , DOI: 10.1002/cbic.201900765
Vlastimil Jirasko 1 , Nils-Alexander Lakomek 1 , Susanne Penzel 1 , Marie-Laure Fogeron 2 , Ralf Bartenschlager 3, 4 , Beat H Meier 1 , Anja Böckmann 2
Affiliation  

Proton-detected 100 kHz magic-angle-spinning (MAS) solid-state NMR is an emerging analysis method for proteins with only hundreds of microgram quantities, and thus allows structural investigation of eukaryotic membrane proteins. This is the case for the cell-free synthesized hepatitis C virus (HCV) nonstructural membrane protein 4B (NS4B). We demonstrate NS4B sample optimization using fast reconstitution schemes that enable lipid-environment screening directly by NMR. 2D spectra and relaxation properties guide the choice of the best sample preparation to record 2D 1 H-detected 1 H,15 N and 3D 1 H,13 C,15 N correlation experiments with linewidths and sensitivity suitable to initiate sequential assignments. Amino-acid-selectively labeled NS4B can be readily obtained using cell-free synthesis, opening the door to combinatorial labeling approaches which should enable structural studies.

中文翻译:

来自丙型肝炎病毒的无细胞合成 α-螺旋跨膜蛋白 NS4B 的质子检测固态 NMR。

质子检测 100 kHz 魔角自旋 (MAS) 固态 NMR 是一种新兴的分析方法,适用于仅数百微克的蛋白质,因此可以对真核膜蛋白进行结构研究。无细胞合成的丙型肝炎病毒 (HCV) 非结构膜蛋白 4B (NS4B) 就是这种情况。我们使用快速重构方案演示了 NS4B 样品优化,该方案能够直接通过 NMR 进行脂质环境筛选。2D 光谱和弛豫特性指导选择最佳样品制备,以记录 2D 1 H 检测的 1 H, 15 N 和 3D 1 H, 13 C, 15 N 相关实验,其线宽和灵敏度适合启动顺序分配。使用无细胞合成可以很容易地获得氨基酸选择性标记的 NS4B,这为组合标记方法打开了大门,从而可以进行结构研究。
更新日期:2019-12-18
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