Integral membrane protein structure determination using pseudocontact shifts.
Integral membrane protein structure determination using pseudocontact shifts.
复制标题
使用伪接触位移测定整体膜蛋白结构。
DOI：
10.1007/s10858-015-9899-6
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发表时间：
2015
影响因子：
2.7
通讯作者：
Crick DJ
中科院分区：
文献类型：
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作者：
Crick DJ
获得足够的实验约束可能是较大蛋白质的NMR结构测定的限制因素。对于大的组装体，例如已经在膜模拟环境中溶解的膜蛋白，情况尤其如此。虽然在这种情况下，广泛的氘化策略经常使用的目的是提高光谱质量，这些计划往往限制了NOE的数量，使互补策略非常有利于成功的结构解析。最近，镧系元素诱导的伪接触位移（PCS）已被确立为球状蛋白质的结构工具。在这里，我们证明了基于PCS的方法可以成功地应用于膜蛋白的结构测定。使用7 TM α-螺旋微生物受体pSRII，我们表明，PCS衍生的限制镧系元素结合标签连接到四个不同的位置的蛋白质促进骨架结构的确定时，结合有限的NOE。相反，同一组NOE无法确定正确的3D折叠。后一种情况在多局部α-螺旋膜蛋白中经常遇到，因此PCS方法甚至适用于这类特别具有挑战性的膜蛋白。测量PCSs的容易性使得这对于一般的大膜蛋白的结构测定是有吸引力的途径。
Obtaining enough experimental restraints can be a limiting factor in the NMR structure determination of larger proteins. This is particularly the case for large assemblies such as membrane proteins that have been solubilized in a membrane-mimicking environment. Whilst in such cases extensive deuteration strategies are regularly utilised with the aim to improve the spectral quality, these schemes often limit the number of NOEs obtainable, making complementary strategies highly beneficial for successful structure elucidation. Recently, lanthanide-induced pseudocontact shifts (PCSs) have been established as a structural tool for globular proteins. Here, we demonstrate that a PCS-based approach can be successfully applied for the structure determination of integral membrane proteins. Using the 7TM α-helical microbial receptor pSRII, we show that PCS-derived restraints from lanthanide binding tags attached to four different positions of the protein facilitate the backbone structure determination when combined with a limited set of NOEs. In contrast, the same set of NOEs fails to determine the correct 3D fold. The latter situation is frequently encountered in polytopical α-helical membrane proteins and a PCS approach is thus suitable even for this particularly challenging class of membrane proteins. The ease of measuring PCSs makes this an attractive route for structure determination of large membrane proteins in general.