Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston Massachusetts 02115, USA.
J Am Chem Soc. 2009 Sep 16;131(36):12970-8. doi: 10.1021/ja902012x.
Four-dimensional (4D) NOESY spectra provide unambiguous distance information at a resolution that cannot be achieved in fewer dimensions and thus increase the quality of biomolecular structure determination substantially. Since the degree of chemical shift degeneracy increases with protein size, the use of 4D NOESY spectra is particularly important for large proteins. The potential high resolution in 4D spectra cannot be achieved in a reasonable time with conventional acquisition routines that sample the Nyquist grid uniformly. It can, however, be obtained with nonuniform sampling of the data grid, but optimal processing of such data has not yet been established. Here we describe a processing method for a pair of sparsely sampled 4D NOESY spectra, a methyl-methyl and an amide-methyl NOESY, recorded on a perdeuterated protein with protonated isoleucine, leucine, and valine methyl groups. The coupled multidimensional decomposition (Co-MDD) of these two spectra together with a 2D template spectrum results in a substantial increase in sensitivity, evidenced by 50-100% additional cross peaks, when compared to alternative processing schemes. At the same time, Co-MDD allows the use of low sparse levels of 10-15% of the full data grid for NOESY spectra. For the 283-residue integral human membrane protein VDAC-1, which has a rotational correlation time of about 70 ns in detergent micelles, the two 4D Co-MDD NOESYs yielded a total of 366 NOEs, resulting in 139 unambiguous upper limit distance constraints for the structure calculation.
四维（4D）NOESY 谱在分辨率上提供了明确的距离信息，这种分辨率是在更少的维度下无法实现的，因此大大提高了生物分子结构测定的质量。由于化学位移简并度随蛋白质大小的增加而增加，因此 4D NOESY 谱的使用对于大蛋白质尤其重要。在常规的以奈奎斯特网格均匀采样的采集例程中，4D 谱的潜在高分辨率无法在合理的时间内实现。然而，可以通过对数据网格进行非均匀采样来实现，但尚未建立这种数据的最佳处理方法。在这里，我们描述了一种处理一对稀疏采样的 4D NOESY 谱的方法，即甲基-甲基和酰胺-甲基 NOESY，这些谱是在氘代蛋白上记录的，其中质子化的异亮氨酸、亮氨酸和缬氨酸甲基。这两个谱与 2D 模板谱的耦合多维分解（Co-MDD）导致灵敏度显著增加，与替代处理方案相比，额外的交叉峰增加了 50-100%。同时，Co-MDD 允许在 NOESY 谱中使用低稀疏水平的 10-15%全数据网格。对于 283 个残基的完整人膜蛋白 VDAC-1，其在去污剂胶束中的旋转相关时间约为 70 ns，两个 4D Co-MDD NOESY 总共产生了 366 个 NOE，从而为结构计算产生了 139 个明确的上限距离约束。
