A novel tool for probing membrane protein structure: Solid-state NMR with proton spin diffusion and X-nucleus detection
A novel tool for probing membrane protein structure: Solid-state NMR with proton spin diffusion and X-nucleus detection
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DOI：
10.1021/ja972655e
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发表时间：
1998-05-27
影响因子：
15
通讯作者：
Thompson, LK
中科院分区：
文献类型：
--
作者：
Kumashiro, KK;Schmidt-Rohr, K;Thompson, LK
我们提出了一种新的固态NMR方法，基于H-1自旋扩散与X-核（N-15，C-13，P-31）检测，用于研究膜蛋白的结构。对于在X-核光谱中具有可分辨信号的任何片段，可以确定插入脂质双层的深度。该技术代表了Goldman-Shen H-1自旋扩散实验与X-核检测在240 K下凝胶状态的水合脂质双层（>25%水重量）中的蛋白质的适应性。实验表明，21-kDa的通道形成结构域的毒素样大肠杆菌素E1分子纳入脂质囊泡。在我们的样品中超过32%的质子是在移动的H2O分子，这可以有效地选择由H-1 T-2过滤器中的Goldman-Shen序列。从移动的H2O的H-1磁化的大肠杆菌素E1通道域的转移是80%完成，只有5毫秒。这种转移到蛋白质，探测的酰胺N-15信号，是比转移到刚性质子平均更快，证明了大部分的蛋白质是优先位于水和脂质双层之间。从自旋扩散和偶极退相数据，60%的24个赖氨酸侧基被证明是高度移动的。使用脂质磷酸盐头基中的P-31和脂质酰基链中的C-13核作为自旋扩散的距离标记物，证明了定量深度分析。
We present a new solid-state NMR approach, based on H-1 spin diffusion with X-nucleus (N-15, C-13, P-31) detection, for investigating the structure of membrane proteins. For any segment with a resolvable signal in the X-nucleus spectrum, the depth of insertion into the lipid bilayer can be determined. The technique represents the adaptation of the Goldman-Shen H-1 spin-diffusion experiment with X-nucleus detection to proteins in hydrated Lipid bilayers (>25% water by weight)in the gel state at 240 K. The experiments are demonstrated on the 21-kDa channel-forming domain of the toxin-like colicin E1 molecule incorporated into lipid vesicles. More than 32% of the protons in our sample are in mobile H2O molecules, which can be selected efficiently by the H-1 T-2 filter in the Goldman-Shen sequence. The transfer of H-1 magnetization from mobile H2O to the colicin E1 channel domain is 80% complete within only 5 ms. This transfer to the protein, probed by the amide N-15 signals, is faster than the transfer to the rigid protons on average, proving that most of the protein is preferentially located between the water and the lipid bilayer. From the spin-diffusion and dipolar-dephasing data, 60% of the 24 lysine side groups are shown to be highly mobile. Quantitative depth profiling is demonstrated using the P-31 in the Lipid phosphate head groups and the C-13 nuclei in the Lipid acyl chains as distance markers for the spin diffusion.