Chemical shift assignment of the transmembrane helices of DsbB, a 20-kDa integral membrane enzyme, by 3D magic-angle spinning NMR spectroscopy
Chemical shift assignment of the transmembrane helices of DsbB, a 20-kDa integral membrane enzyme, by 3D magic-angle spinning NMR spectroscopy
复制标题
DOI：
10.1110/ps.073225008
复制
发表时间：
2008-02-01
期刊：
影响因子：
8
通讯作者：
Rienstra, Chad M.
中科院分区：
文献类型：
--
作者：
Li, Ying;Berthold, Deborah A.;Rienstra, Chad M.
大肠杆菌内膜酶DsbB通过将电子从DsbA转移到泛醌来催化周质蛋白中的二硫键形成，泛醌进而直接氧化底物蛋白中的半胱氨酸。我们以前已经表明，DsbB可以在一个状态下，提供高分辨率的魔角旋转（MAS）NMR光谱制备。在这里，我们报告连续的C-13和N-15化学位移分配的跨膜螺旋中的大多数残基，实现了三维（3D）相关实验上均匀的C-13，N-15标记的样品在750 MHz的H-1频率。我们还提出了一个四维（4D）相关谱，这证实了在一些高度拥挤的区域的3D光谱的分配。总体而言，我们的研究结果表明，有可能分配较大的膜蛋白，使用3D和4D相关实验，并形成进一步的结构和动力学研究的DsbB的MAS NMR的基础。
The Escherichia coli inner membrane enzyme DsbB catalyzes disulfide bond formation in periplasmic proteins, by transferring electrons to ubiquinone from DsbA, which in turn directly oxidizes cysteines in substrate proteins. We have previously shown that DsbB can be prepared in a state that gives highly resolved magic-angle spinning (MAS) NMR spectra. Here we report sequential C-13 and N-15 chemical shift assignments for the majority of the residues in the transmembrane helices, achieved by three-dimensional (3D) correlation experiments on a uniformly C-13, N-15-labeled sample at 750-MHz H-1 frequency. We also present a four-dimensional (4D) correlation spectrum, which confirms assignments in some highly congested regions of the 3D spectra. Overall, our results show the potential to assign larger membrane proteins using 3D and 4D correlation experiments and form the basis of further structural and dynamical studies of DsbB by MAS NMR.