Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosome-nascent chain complexes.
Increasing the sensitivity of NMR diffusion measurements by paramagnetic longitudinal relaxation enhancement, with application to ribosome-nascent chain complexes.
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DOI：
10.1007/s10858-015-9968-x
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发表时间：
2015-10
影响因子：
2.7
通讯作者：
Christodoulou J
中科院分区：
文献类型：
--
作者：
Chan SHS;Waudby CA;Cassaignau AME;Cabrita LD;Christodoulou J
大分子的平移扩散可以通过刺激回波(STE)核磁共振实验非侵入性地检测，从而准确地确定其分子大小。这些测量可以成为分子间相互作用和蛋白质折叠和展开的重要探针，并在监测大分子组件(如核糖体-新生链状复合体(RNC))的完整性方面至关重要。然而，这些配合物的核磁共振研究可能会受到它们缓慢滚动、低溶解度(最大浓度高达10μM)和寿命短(导致信号较弱)的严重限制，因此继续提高实验灵敏度是至关重要的。在这里，我们探索了顺磁纵向弛豫增强(PLRE)试剂NiDO2A对15N XSTE灵敏度的影响和肮脏的异核STE实验，这些实验可以用来监测这些不稳定络合物的完整性。我们利用PLRE效应对旋磁比和电子弛豫时间的依赖关系来加速1H磁化强度的恢复，而不会在扩散延迟期间对NZ上的存储造成不利影响，也不会引起显著的横向弛豫线加宽。通过将纵向松弛优化的SOLID脉冲序列与NiDO2A一起应用到70年代的大肠杆菌核糖体和RNC中，在不影响样品完整性的情况下，与XSTE相比，核磁共振扩散灵敏度提高了4.5倍，二维核磁共振灵敏度提高了约1.9倍。我们预计，这些结果将极大地推动核磁共振技术在探索核糖体动态区域和其他大的、不稳定的大分子组装方面的应用。
The translational diffusion of macromolecules can be examined non-invasively by stimulated echo (STE) NMR experiments to accurately determine their molecular sizes. These measurements can be important probes of intermolecular interactions and protein folding and unfolding, and are crucial in monitoring the integrity of large macromolecular assemblies such as ribosome–nascent chain complexes (RNCs). However, NMR studies of these complexes can be severely constrained by their slow tumbling, low solubility (with maximum concentrations of up to 10 μM), and short lifetimes resulting in weak signal, and therefore continuing improvements in experimental sensitivity are essential. Here we explore the use of the paramagnetic longitudinal relaxation enhancement (PLRE) agent NiDO2A on the sensitivity of 15N XSTE and SORDID heteronuclear STE experiments, which can be used to monitor the integrity of these unstable complexes. We exploit the dependence of the PLRE effect on the gyromagnetic ratio and electronic relaxation time to accelerate recovery of 1H magnetization without adversely affecting storage on Nz during diffusion delays or introducing significant transverse relaxation line broadening. By applying the longitudinal relaxation-optimized SORDID pulse sequence together with NiDO2A to 70S Escherichia coli ribosomes and RNCs, NMR diffusion sensitivity enhancements of up to 4.5-fold relative to XSTE are achieved, alongside ~1.9-fold improvements in two-dimensional NMR sensitivity, without compromising the sample integrity. We anticipate these results will significantly advance the use of NMR to probe dynamic regions of ribosomes and other large, unstable macromolecular assemblies.