Structural and orientational constraints of bacteriorhodopsin in purple membranes determined by oriented-sample solid-state NMR spectroscopy
Structural and orientational constraints of bacteriorhodopsin in purple membranes determined by oriented-sample solid-state NMR spectroscopy
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DOI：
10.1016/j.jsb.2004.10.002
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发表时间：
2005-01-01
影响因子：
3
通讯作者：
Watts, A
中科院分区：
文献类型：
--
作者：
Kamihira, M;Vosegaard, T;Watts, A
我们报告的第一次，定向样品固态NMR实验，特别是极化反转自旋交换在魔角（PISEMA）和H-1-N-15异源化学位移相关（HETCOR），适用于一个完整的七跨膜蛋白，细菌视紫红质（bR），在自然膜。[N-15]Met-bR的光谱揭示了螺旋和环区域的明显可区分的信号。通过螺旋共振的解卷积，可以建立对一些螺旋倾斜角的约束。据估计，螺旋B的细胞外部分与正常膜的倾斜度小于5。而螺旋A的倾斜度估计为18- 22度，两者都与大多数晶体结构一致。实验PISEMA光谱与基于晶体结构的模拟光谱的比较表明，PISEMA和HETCOR实验是非常敏感的多面体蛋白质结构，和固态NMR光谱膜嵌入bR匹配最有利的最近1FBB电子晶体学结构。这些结果表明，这种方法有可能产生的结构和取向的限制，同时插入和功能上有能力在一个天然的膜大的整体多面体蛋白质。(C)2004爱思唯尔公司All rights reserved.
We report for the first time, oriented-sample solid-state NMR experiments, specifically polarization inversion spin exchange at the magic angle (PISEMA) and H-1-N-15 heteronuclear chemical shift correlation (HETCOR), applied to an integral seven-transmembrane protein, bacteriorhodopsin (bR), in natural membranes. The spectra of [N-15]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 from the membrane normal. while the tilt of helix A was estimated to be 18-22degrees, 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. (C) 2004 Elsevier Inc. All rights reserved.