Kamihira Miya, Vosegaard Thomas, Mason A James, Straus Suzana K, Nielsen Niels Chr, Watts Anthony
Biomembrane Structure Unit, Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, United Kingdom.
J Struct Biol. 2005 Jan;149(1):7-16. doi: 10.1016/j.jsb.2004.10.002.
We report for the first time, oriented-sample solid-state NMR experiments, specifically polarization inversion spin exchange at the magic angle (PISEMA) and 1H-15N heteronuclear chemical shift correlation (HETCOR), applied to an integral seven-transmembrane protein, bacteriorhodopsin (bR), in natural membranes. The spectra of [15N]Met-bR revealed clearly distinguishable signals from the helical and loop regions. By deconvolution of the helix resonances, it was possible to establish constraints for some helix tilt angles. It was estimated that the extracellular section of helix B has a tilt of less than 5 degrees from the membrane normal, while the tilt of helix A was estimated to be 18-22 degrees , both of which are in agreement with most crystal structures. Comparison of the experimental PISEMA spectrum with simulated spectra based on crystal structures showed that PISEMA and HETCOR experiments are extremely sensitive to the polytopic protein structure, and the solid-state NMR spectra for membrane-embedded bR matched most favorably with the recent 1FBB electron crystallography structure. These results suggest that this approach has the potential to yield structural and orientational constraints for large integral polytopic proteins whilst intercalated and functionally competent in a natural membrane.
我们首次报道了将取向样品固态核磁共振实验，特别是魔角极化反转自旋交换（PISEMA）和1H-15N异核化学位移相关（HETCOR），应用于天然膜中完整的七跨膜蛋白细菌视紫红质（bR）。[15N]Met-bR的光谱清楚地显示出来自螺旋区和环区的可区分信号。通过对螺旋共振进行反卷积，可以确定一些螺旋倾斜角的限制条件。据估计，螺旋B的细胞外部分与膜法线的倾斜度小于5度，而螺旋A的倾斜度估计为18 - 22度，这两者都与大多数晶体结构一致。将实验性的PISEMA光谱与基于晶体结构的模拟光谱进行比较表明，PISEMA和HETCOR实验对多结构域蛋白结构极其敏感，并且膜嵌入bR的固态核磁共振光谱与最近的1FBB电子晶体学结构最为匹配。这些结果表明，这种方法有潜力为嵌入天然膜且功能正常的大型完整多结构域蛋白提供结构和取向限制条件。
