Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.
Protein Sci. 2012 Apr;21(4):589-95. doi: 10.1002/pro.2038. Epub 2012 Mar 2.
NMR paramagnetic relaxation enhancement (PRE) provides long-range distance constraints (~15-25 Å) that can be critical to determining overall protein topology, especially where long-range NOE information is unavailable such as in the case of larger proteins that require deuteration. However, several challenges currently limit the use of NMR PRE for α-helical membrane proteins. One challenge is the nonspecific association of the nitroxide spin label to the protein-detergent complex that can result in spurious PRE derived distance restraints. The effect of the nitroxide spin label contaminant is evaluated and quantified and a robust method for the removal of the contaminant is provided to advance the application of PRE restraints to membrane protein NMR structure determination.
NMR 顺磁各向异性弛豫增强(PRE)提供了长程距离约束(约 15-25Å),这对于确定整体蛋白质拓扑结构至关重要,尤其是在无法获得长程 NOE 信息的情况下,例如对于需要氘代的较大蛋白质。然而,目前有几个挑战限制了 NMR PRE 在 α-螺旋膜蛋白中的应用。一个挑战是氮氧自由基自旋标记物与蛋白质-去污剂复合物的非特异性结合,这可能导致虚假的 PRE 衍生距离约束。评估和量化了氮氧自由基自旋标记物污染物的影响,并提供了一种可靠的去除污染物的方法,以推进 PRE 约束在膜蛋白 NMR 结构测定中的应用。