UMR 7099, CNRS and Université Paris Diderot, IBPC, 13 rue Pierre et Marie Curie, F-75005 Paris, France.
J Magn Reson. 2010 May;204(1):155-9. doi: 10.1016/j.jmr.2010.02.003. Epub 2010 Feb 11.
High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75kDa pentameric alpha-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR.
迄今为止，大多数膜蛋白的高分辨率结构都是通过 X 射线晶体学获得的，这些蛋白质的样本是被去污剂包围的。固态 NMR 的最新发展为研究膜内的完整膜蛋白开辟了一条新途径。与此同时，无细胞表达技术的扩展使得可以生产专门用于 NMR 的膜蛋白。我们在这里介绍了一种通过无细胞表达选择性标记的膜蛋白的原位固态 NMR 研究。该样品由 MscL（大电导机械敏感通道）组成，MscL 是一种来自大肠杆菌的 75kDa 五聚体α螺旋离子通道，在水合脂质双层中重新构建。与均匀标记的蛋白质样品相比，无细胞表达的蛋白质样品中的光谱拥挤度大大降低。这种方法可能是通过固态 NMR 对膜蛋白进行光谱分配和结构确定所需的关键步骤。
