Site-directed parallel spin-labeling and paramagnetic relaxation enhancement in structure determination of membrane proteins by solution NMR spectroscopy
Site-directed parallel spin-labeling and paramagnetic relaxation enhancement in structure determination of membrane proteins by solution NMR spectroscopy
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DOI：
10.1021/ja0574825
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发表时间：
2006-04-05
影响因子：
15
通讯作者：
Tamm, LK
中科院分区：
文献类型：
--
作者：
Liang, BY;Bushweller, JH;Tamm, LK
一个主要的挑战，通过溶液NMR光谱的结构测定的完整的膜蛋白是有限的NOE限制在这些系统中源于广泛的氘代。顺磁弛豫增强（PRE）通过氮氧自由基自旋标签可以提供有价值的远程距离的信息，但在实践中，有限制，在其应用于膜蛋白，因为自旋标签往往不完全减少在高度非极性的环境。使用完整的膜蛋白OmpA作为模型系统，我们介绍了一种平行的自旋标记的方法，顺磁性和反磁性标签，并显示在15-24埃的范围内的距离可以很容易地确定。在11个水暴露和脂质覆盖的网站，并测量320 PRE距离限制的蛋白质进行标记。这些限制的加入导致了OmpA的计算骨架结构的显着改善。合理质量的结构甚至可以仅用PRE距离约束来计算，即，在没有NOE距离限制的情况下。
A major challenge for the structure determination of integral membrane proteins by solution NMR spectroscopy is the limited number of NOE restraints in these systems stemming from extensive deuteration. Paramagnetic relaxation enhancement (PRE) by means of nitroxide spin-labels can provide valuable long-range distance information but, in practice, has limits in its application to membrane proteins because spin-labels are often incompletely reduced in highly apolar environments. Using the integral membrane protein OmpA as a model system, we introduce a method of parallel spin-labeling with paramagnetic and diamagnetic labels and show that distances in the range 15-24 angstrom can be readily determined. The protein was labeled at 11 water-exposed and lipid-covered sites, and 320 PRE distance restraints were measured. The addition of these restraints resulted in significant improvement of the calculated backbone structure of OmpA. Structures of reasonable quality can even be calculated with PRE distance restraints only, i.e., in the absence of NOE distance restraints.