Rovnyak David, Frueh Dominique P, Sastry Mallika, Sun Zhen-Yu J, Stern Alan S, Hoch Jeffrey C, Wagner Gerhard
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
J Magn Reson. 2004 Sep;170(1):15-21. doi: 10.1016/j.jmr.2004.05.016.
Non-uniform sampling is shown to provide significant time savings in the acquisition of a suite of three-dimensional NMR experiments utilized for obtaining backbone assignments of H, N, C', CA, and CB nuclei in proteins : HNCO, HN(CA)CO, HNCA, HN(CO)CA, HNCACB, and HN(CO)CACB. Non-uniform sampling means that data were collected for only a subset of all incremented evolution periods, according to a user-specified sampling schedule. When the suite of six 3D experiments was acquired in a uniform fashion for an 11 kDa cytoplasmic domain of a membrane protein at 1.5 mM concentration, a total of 146 h was consumed. With non-uniform sampling, the same experiments were acquired in 32 h and, through subsequent maximum entropy reconstruction, yielded spectra of similar quality to those obtained by conventional Fourier transform of the uniformly acquired data. The experimental time saved with this methodology can significantly accelerate protein structure determination by NMR, particularly when combined with the use of automated assignment software, and enable the study of samples with poor stability at room temperature. Since it is also possible to use the time savings to acquire a greater numbers of scans to increase sensitivity while maintaining high resolution, this methodology will help extend the size limit of proteins accessible to NMR studies, and open the way to studies of samples that suffer from solubility problems.
结果表明，非均匀采样在获取用于确定蛋白质中H、N、C′、CA和CB原子核主链归属的一组三维核磁共振实验时，能显著节省时间：HNCO、HN(CA)CO、HNCA、HN(CO)CA、HNCACB和HN(CO)CACB。非均匀采样意味着根据用户指定的采样计划，仅对所有递增演化期的一个子集收集数据。当以均匀方式对浓度为1.5 mM的膜蛋白11 kDa细胞质结构域进行这组六个三维实验时，总共耗时146小时。采用非均匀采样时，相同的实验在32小时内完成，并且通过随后的最大熵重建，得到的谱图质量与通过对均匀采集的数据进行传统傅里叶变换得到的谱图相似。这种方法节省的实验时间可以显著加快通过核磁共振确定蛋白质结构的速度，特别是与使用自动归属软件相结合时，还能对室温下稳定性较差的样品进行研究。由于也可以利用节省的时间进行更多次数的扫描以提高灵敏度同时保持高分辨率，这种方法将有助于扩大可通过核磁共振研究的蛋白质的尺寸限制，并为研究存在溶解性问题的样品开辟道路。
