Solid-State NMR Spectra of Lipid-Anchored Proteins under Magic Angle Spinning
Solid-State NMR Spectra of Lipid-Anchored Proteins under Magic Angle Spinning
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DOI：
10.1021/jp4124106
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发表时间：
2014-03-06
影响因子：
3.3
通讯作者：
Shimamoto, Keiko
中科院分区：
文献类型：
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作者：
Nomura, Kaoru;Harada, Erisa;Shimamoto, Keiko
固体核磁共振是一种很有前途的工具，用于阐明膜相关的生物现象。利用魔角自旋（MAS）技术对包埋在脂双层中的脂锚定蛋白进行了高分辨固体核磁共振谱的测量。到目前为止，固态NMR测量的脂质锚定蛋白尚未完成，由于难以提供足够量的稳定同位素标记的样品中的脂质锚定蛋白的过表达需要复杂的翻译后修饰。我们设计了一个假脂质锚定蛋白，其中蛋白组分在E.大肠杆菌中，并连接到化学合成的脂质锚模拟物。使用两种类型的膜，脂质体和bicelles，我们证明了不同类型的插入程序的脂质锚定蛋白到膜。在脂质体样品中，我们能够在MAS下观察到交叉极化和C-13-C-13化学位移相关光谱，表明脂质体样品可以用于使用基于偶极的NMR实验分析分子相互作用。相比之下，双胞样品通过基于标量的实验显示出足够的光谱质量。弛豫时间和蛋白质膜相互作用能够在双胞样品中进行分析。这些结果表明，两种类型的样品系统的适用性，阐明的作用，脂质锚在调节不同的生物现象。
Solid-state NMR is a promising tool for elucidating membrane-related biological phenomena. We achieved the measurement of high-resolution solid-state NMR spectra for a lipid-anchored protein embedded in lipid bilayers under magic angle spinning (MAS). To date, solid-state NMR measurements of lipid-anchored proteins have not been accomplished due to the difficulty in supplying sufficient amount of stable isotope labeled samples in the overexpression of lipid-anchored proteins requiring complex posttranslational modification. We designed a pseudo lipid-anchored protein in which the protein component was expressed in E. coli and attached to a chemically synthesized lipid-anchor mimic. Using two types of membranes, liposomes and bicelles, we demonstrated different types of insertion procedures for lipid-anchored protein into membranes. In the liposome sample, we were able to observe the cross-polarization and the C-13-C-13 chemical shift correlation spectra under MAS, indicating that the liposome sample can be used to analyze molecular interactions using dipolar-based NMR experiments. In contrast, the bicelle sample showed sufficient quality of spectra through scalar-based experiments. The relaxation times and protein membrane interaction were capable of being analyzed in the bicelle sample. These results demonstrated the applicability of two types of sample system to elucidate the roles of lipid-anchors in regulating diverse biological phenomena.