NMR Spectroscopy of Soluble Protein Complexes at One Mega-Dalton and Beyond
NMR Spectroscopy of Soluble Protein Complexes at One Mega-Dalton and Beyond
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DOI：
10.1002/anie.201301215
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发表时间：
2013-08-12
影响因子：
16.6
通讯作者：
Reif, Bernd
中科院分区：
文献类型：
--
作者：
Mainz, Andi;Religa, Tomasz L.;Reif, Bernd
溶液状态核磁共振（NMR）光谱是一种非常成功的方法来表征生物分子在溶液中的结构和动力学。[1，2]然而，随着分子量的增加，共振线显着变宽，同时影响灵敏度和分辨率。这种加宽是由于长的旋转翻滚相关时间tc，这增强了横向弛豫。[3]通过使用横向驰豫优化光谱（TROSY）可以部分避免有害的驰豫效应，该光谱基于不同驰豫途径的相互抵消，并且允许选择缓慢驰豫的多重峰成分。[4，5]因此，TROSY型技术能够表征超分子组装体，如GroEL-GroES，[6] p53/Hsp 90，[7] ATP酶马达SecA，[8] ClpP，[9]或20 S蛋白酶体。[10然而，即使在非常高的磁场下，低分子翻滚速率也限制了溶液状态NMR的适用性。特别是，超过80 kDa的蛋白质复合物的骨架共振的分配变得越来越困难。通常情况下，甲基为基础的实验进行非常大的系统，因为整体翻滚的贡献减少，由于快速的三倍旋转的甲基。[5]In魔角旋转（MAS）固态NMR中，固定的样品在圆柱形转子中快速旋转，该转子相对于NMR光谱仪的磁场以54.748 °的角度VMA倾斜。[12]MAS通过重新聚焦相干各向异性相互作用（例如偶极耦合和化学位移各向异性）产生共振线变窄。
Solution-state nuclear magnetic resonance (NMR) spectroscopy is a very successful method to characterize the structure and dynamics of biomolecules in solution.[1, 2] However, resonance lines broaden significantly with increasing molecular weight, simultaneously affecting sensitivity and resolution. This broadening is due to long rotational tumbling correlation times tc, which enhance transverse relaxation.[3] Deleterious relaxation effects can be partially avoided by the use of transverse relaxation-optimized spectroscopy (TROSY), which is based on mutual cancellation of different relaxation pathways and which allows the selection of slowly relaxing multiplet components.[4, 5] TROSY-type techniques have thus enabled the characterization of supramolecular assemblies like GroEL-GroES,[6] p53/Hsp90,[7] the ATPase motor SecA,[8] ClpP,[9] or the 20S proteasome.[10, 11] However, even at very high magnetic fields, low molecular tumbling rates limit the applicability of solution-state NMR. In particular, the assignment of backbone resonances becomes increasingly difficult for protein complexes beyond 80 kDa. Typically, methyl-based experiments are carried out for very large systems, as contributions from overall tumbling are reduced owing to the fast three-fold rotation of the methyl group.[5]In magic-angle spinning (MAS) solid-state NMR, immobilized samples are spun rapidly in a cylindrical rotor, which is inclined at an angle VMA of 54.748 relative to the magnetic field of the NMR spectrometer.[12] MAS yields resonance line narrowing by refocusing coherent anisotropic interactions, such as the dipolar coupling and the chemical shift anisotropy.