Frericks Heather L, Zhou Donghua H, Yap Lai Lai, Gennis Robert B, Rienstra Chad M
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
J Biomol NMR. 2006 Sep;36(1):55-71. doi: 10.1007/s10858-006-9070-5. Epub 2006 Sep 9.
Recent progress in magic-angle spinning (MAS) solid-state NMR (SSNMR) has enabled multidimensional studies of large, macroscopically unoriented membrane proteins with associated lipids, without the requirement of solubility that limits other structural techniques. Here we present initial sample preparation and SSNMR studies of a 144 kDa integral membrane protein, E. coli cytochrome bo(3) oxidase. The optimized protocol for expression and purification yields approximately 5 mg of the enzymatically active, uniformly (13)C,(15)N-enriched membrane protein complex from each liter of growth medium. The preparation retains endogenous lipids and yields spectra of high sensitivity and resolution, consistent with a folded, homogenous protein. Line widths of isolated signals are less than 0.5 ppm, with a large number of individual resonances resolved in the 2D and 3D spectra. The (13)C chemical shifts, assigned by amino acid type, are consistent with the secondary structure previously observed by diffraction methods. Although the structure is predominantly helical, the percentage of non-helical signals varies among residue types; these percentages agree well between the NMR and diffraction data. Samples show minimal evidence of degradation after several weeks of NMR data acquisition. Use of a triple resonance scroll resonator probe further improves sample stability and enables higher power decoupling, higher duty cycles and more advanced 3D experiments to be performed. These initial results in cytochrome bo(3) oxidase demonstrate that multidimensional MAS SSNMR techniques have sufficient sensitivity and resolution to interrogate selected parts of a very large uniformly (13)C,(15)N-labeled membrane protein.
魔角旋转（MAS）固态核磁共振（SSNMR）技术的最新进展使得对与脂质相关的大型、宏观上无定向的膜蛋白进行多维研究成为可能，而无需溶解性，这限制了其他结构技术。在此，我们展示了对一种144 kDa的整合膜蛋白——大肠杆菌细胞色素bo(3)氧化酶的初步样品制备和SSNMR研究。用于表达和纯化的优化方案每升生长培养基可产生约5 mg具有酶活性、均匀（13）C、（15）N富集的膜蛋白复合物。该制备方法保留了内源性脂质，并产生了高灵敏度和分辨率的光谱，与折叠的、均匀的蛋白质一致。孤立信号的线宽小于0.5 ppm，在二维和三维光谱中分辨出大量单个共振峰。按氨基酸类型归属的（13）C化学位移与先前通过衍射方法观察到的二级结构一致。尽管结构主要是螺旋结构，但非螺旋信号的百分比在不同残基类型中有所变化；这些百分比在核磁共振和衍射数据之间吻合得很好。在进行数周的核磁共振数据采集后，样品显示出最小程度的降解迹象。使用三重共振涡旋谐振器探头进一步提高了样品稳定性，并能够进行更高功率的去耦、更高的占空比以及更先进的三维实验。细胞色素bo(3)氧化酶的这些初步结果表明，多维MAS SSNMR技术具有足够的灵敏度和分辨率来研究非常大的均匀（13）C、（15）N标记膜蛋白的选定部分。
