Xia Youlin, Zhu Guang, Veeraraghavan Sudha, Gao Xiaolian
Department of Chemistry, University of Houston, Houston, TX 77004-5003, U.S.A.
J Biomol NMR. 2004 Aug;29(4):467-76. doi: 10.1023/B:JNMR.0000034352.75619.3f.
High throughput structure determination of proteins will contribute to the success of proteomics investigations. The G-Matrix Fourier Transformation NMR (GFT-NMR) method significantly shortens experimental time by reducing the number of the dimensions of data acquisition for isotopically labeled proteins (Kim, S. and Szyperski, T. (2003) J. Am. Chem. Soc. 125, 1385). We demonstrate herein a suite of ten 3D-->2D or (3,2)D GFT-NMR experiments using (13)C/(15)N-labeled ubiquitin. These experiments were completed within 18 hours, representing a 4- to 18-fold reduction in data acquisition time compared to the corresponding conventional 3D experiments. A subset of the GFT-NMR experiments, (3,2)D HNCO, HNCACB, HN(CO)CACB, and 2D (1)H-(15)N HSQC, which are necessary for backbone assignments, were carried out within 6 hours. To facilitate the analysis of the GFT-NMR spectra, we developed automated procedures for viewing and analyzing the GFT-NMR spectra. Our overall strategy allows (3,2)D GFT-NMR experiments to be readily performed and analyzed. Nevertheless, the increase in spectral overlap and the reduction in signal sensitivity in these fast NMR experiments presently limit their application to relatively small proteins.
蛋白质的高通量结构测定将有助于蛋白质组学研究的成功。G-矩阵傅里叶变换核磁共振（GFT-NMR）方法通过减少同位素标记蛋白质数据采集的维度数量，显著缩短了实验时间（Kim, S. 和 Szyperski, T. (2003) J. Am. Chem. Soc. 125, 1385）。我们在此展示了一套使用（13）C/（15）N标记的泛素进行的十个3D→2D或（3,2）D GFT-NMR实验。这些实验在18小时内完成，与相应的传统3D实验相比，数据采集时间减少了4至18倍。对于主链归属必需的GFT-NMR实验的一个子集，即（3,2）D HNCO、HNCACB、HN(CO)CACB和2D （1）H-（15）N HSQC，在6小时内完成。为便于分析GFT-NMR谱图，我们开发了用于查看和分析GFT-NMR谱图的自动化程序。我们的总体策略使（3,2）D GFT-NMR实验能够轻松进行和分析。然而，这些快速核磁共振实验中谱图重叠的增加和信号灵敏度的降低目前限制了它们仅适用于相对较小的蛋白质。
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