NMR observation of substrate in the binding site of an active sugar-H+ symport protein in native membranes.
NMR observation of substrate in the binding site of an active sugar-H+ symport protein in native membranes.
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天然膜中活性糖-H 同向转运蛋白结合位点的底物的 NMR 观察。
DOI：
10.1073/pnas.91.9.3877
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发表时间：
1994
影响因子：
11.1
通讯作者：
Peter J.F. Henderson
中科院分区：
文献类型：
--
作者：
P. Spooner;N. Rutherford;Anthony Watts;Peter J.F. Henderson
用核磁共振方法直接观察了半乳糖-H ~+转运蛋白GalP在其天然环境--大肠杆菌内膜中的底物结合特征。沉淀的内膜囊泡含有GalP蛋白，过表达到总蛋白的50%以上的水平，进行了分析，通过13 C魔角旋转NMR，当在其正常的“流体”状态和与D-[1- 13 C]葡萄糖。使用交叉极化的条件，旨在区分单独的结合底物，它是可以检测到少至250 nmol的底物添加到膜含有约0.5 μ mol（约26毫克）的GalP蛋白。由于流体膜对观察到的核的快速弛豫恢复的运动贡献，以及在这些实验中接近静态场不均匀性的高分辨率响应，因此流体膜的测量灵敏度之所以如此之高是可能的。这种良好的光谱分辨率表明，膜的天然状态呈现出具有高结构均匀性的底物结合环境。GalP蛋白、细胞松弛素B和毛喉素的特异性抑制剂，以及D-半乳糖，但不是L-半乳糖，阻止或抑制13 C标记的葡萄糖底物的检测，证实观察到的信号是由于与GalP蛋白的特异性相互作用。这种特异性底物结合表现出对D-葡萄糖的β-端基异构体的偏好，并且在10（-2）s的时间尺度上确定底物移位是缓慢的。这项工作描述了一个简单的NMR方法，它实现了高灵敏度，选择性和分辨率与复杂的膜蛋白相关的核，并可以与其他NMR方法相结合，以产生额外的结构信息的结合位点的电流传输系统，而不将其从其天然的膜环境。
NMR methods have been adopted to observe directly the characteristics of substrate binding to the galactose-H+ symport protein GalP, in its native environment, the inner membranes of Escherichia coli. Sedimented inner-membrane vesicles containing the GalP protein, overexpressed to levels above 50% of total protein, were analyzed by 13C magic-angle spinning NMR, when in their normal "fluid" state and with incorporated D-[1-13C]glucose. Using conditions of cross-polarization intended to discriminate bound substrate alone, it was possible to detect as little as 250 nmol of substrate added to the membranes containing about 0.5 mumol (approximately 26 mg) of GalP protein. Such high measuring sensitivity was possible from the fluid membranes by virtue of their motional contributions to rapid relaxation recovery of the observed nuclei and due to a high-resolution response that approached the static field inhomogeneity in these experiments. This good spectral resolution showed that the native state of the membranes presents a substrate binding environment with high structural homogeneity. Inhibitors of the GalP protein, cytochalasin B and forskolin, which are specific, and D-galactose, but not L-galactose, prevent or suppress detection of the 13C-labeled glucose substrate, confirming that the observed signal was due to specific interactions with the GalP protein. This specific substrate binding exhibits a preference for the beta-anomer of D-glucose and substrate translocation is determined to be slow, on the 10(-2) s time scale. The work describes a straightforward NMR approach, which achieves high sensitivity, selectivity, and resolution for nuclei associated with complex membrane proteins and which may be combined with other NMR methodologies to yield additional structural information on the binding site for the current transport system without isolating it from its native membrane environment.