Complex Carbohydrate Research Center, The University of Georgia, Athens, GA 30602, USA.
J Magn Reson. 2011 Oct;212(2):289-98. doi: 10.1016/j.jmr.2011.07.007. Epub 2011 Jul 21.
Most multi-dimensional solution NMR experiments connect one dimension to another using coherence transfer steps that involve evolution under scalar couplings. While experiments of this type have been a boon to biomolecular NMR the need to work on ever larger systems pushes the limits of these procedures. Spin relaxation during transfer periods for even the most efficient (15)N-(1)H HSQC experiments can result in more than an order of magnitude loss in sensitivity for molecules in the 100 kDa range. A relatively unexploited approach to preventing signal loss is to avoid coherence transfer steps entirely. Here we describe a scheme for multi-dimensional NMR spectroscopy that relies on direct frequency encoding of a second dimension by multi-frequency decoupling during acquisition, a technique that we call MD-DIRECT. A substantial improvement in sensitivity of (15)N-(1)H correlation spectra is illustrated with application to the 21 kDa ADP ribosylation factor (ARF) labeled with (15)N in all alanine residues. Operation at 4°C mimics observation of a 50 kDa protein at 35°C.
大多数多维溶液 NMR 实验通过涉及标量耦合下演化的相干转移步骤将一维与另一维连接。虽然这类实验对生物分子 NMR 是一个福音，但需要处理越来越大的系统，这推到了这些程序的极限。即使是最有效的 (15)N-(1)H HSQC 实验，在转移期间的自旋弛豫也会导致 100 kDa 范围内的分子灵敏度损失一个数量级以上。一种相对未被充分利用的防止信号损失的方法是完全避免相干转移步骤。在这里，我们描述了一种依赖于在采集过程中通过多频去耦直接对第二维进行频率编码的多维 NMR 光谱方案，我们称之为 MD-DIRECT。通过将其应用于用所有丙氨酸残基标记 (15)N 的 21 kDa ADP 核糖基化因子 (ARF)，(15)N-(1)H 相关光谱的灵敏度得到了显著提高。在 4°C 下操作模拟了在 35°C 下观察 50 kDa 蛋白质的情况。
