Sensitivity enhancement using paramagnetic relaxation in MAS solid-state NMR of perdeuterated proteins
Sensitivity enhancement using paramagnetic relaxation in MAS solid-state NMR of perdeuterated proteins
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DOI：
10.1016/j.jmr.2007.09.007
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发表时间：
2007-12-01
影响因子：
2.2
通讯作者：
Reif, Bernd
中科院分区：
文献类型：
--
作者：
Linser, Rasmus;Chevelkov, Veniamin;Reif, Bernd
此前，石井等人，可以表明螯合的顺磁性离子可以用于显著降低MAS固态NMR实验的再循环延迟[N. P. Wickramasinghe，M. Kotecha，A. Samoson，J. Past，Y.石井，通过使用顺磁金属离子优化H-1 T-1弛豫来增强蛋白质微晶的C-13固态NMR的灵敏度，J. Magn. Reson. 184（2007）350-356]。该方法的应用仅限于非常耐用的样品，其样品稳定性不会受到RF感应加热的影响。此外，由于使用非常短的占空比，在标准MAS PRE实验中探针完整性可能会受到干扰。我们表明，这些有害的影响可以避免，如果采用全氘代蛋白质已从D2 O：H2O = 9：1含有缓冲溶液重结晶。使用鸡α-血影蛋白的SH 3结构域作为模型系统来证明实验。标记方案允许记录质子检测的H-1，N-15相关光谱具有非常高的分辨率，在不存在杂原子偶极去耦。Cu-EDTA作为掺杂剂产生循环延迟的减少高达15倍。特别是，我们发现，1H T-1的散装H-N磁化强度从4.4秒减少到0.3秒，如果Cu-EDTA浓度从0 mM增加到250 mM。可能的扰动，如化学位移的变化或线加宽由于顺磁性螯合物是最小的。在实验过程中未观察到样品降解。(c)2007年爱思唯尔公司All rights reserved.
Previously, Ishii et al., could show that chelated paramagnetic ions can be employed to significantly decrease the recycle delay of a MAS solid-state NMR experiment [N.P. Wickramasinghe, M. Kotecha, A. Samoson, J. Past, Y. Ishii, Sensitivity enhancement in C-13 solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing H-1 T-1 relaxation, J. Magn. Reson. 184 (2007) 350-356]. Application of the method is limited to very robust samples, for which sample stability is not compromised by RF induced heating. In addition, probe integrity might be perturbed in standard MAS PRE experiments due to the use of very short duty cycles. We show that these deleterious effects can be avoided if perdeuterated proteins are employed that have been re-crystallized from D2O:H2O = 9:1 containing buffer solutions. The experiments are demonstrated using the SH3 domain of chicken alpha-spectrin as a model system. The labeling scheme allows to record proton detected H-1, N-15 correlation spectra with very high resolution in the absence of heteronuclear dipolar decoupling. Cu-edta as a doping reagent yields a reduction of the recycle delay by up to a factor of 15. In particular, we find that the 1H T-1 for the bulk H-N magnetization is reduced from 4.4 s to 0.3 s if the Cu-edta concentration is increased from 0 mM to 250 mM. Possible perturbations like chemical shift changes or line broadening due to the paramagnetic chelate complex are minimal. No degradation of our samples was observed in the course of the experiments. (c) 2007 Elsevier Inc. All rights reserved.