Department of Chemical Sciences, Tata Institute of Fundamental Research (TIFR), 1-Homi Bhabha Road, Colaba, Mumbai 400 005, India.
J Magn Reson. 2010 Sep;206(1):134-8. doi: 10.1016/j.jmr.2010.06.013. Epub 2010 Jun 30.
Time-saving in data acquisition is a major thrust of NMR pulse sequence development in the context of structural proteomics research. The conventional HNCA and HN(CA)CO pulse sequences, routinely used for sequential backbone assignment, have the limitation that they cannot distinguish inter- and intra-residue correlations. In order to remove this ambiguity, one has to record HNCO and HN(CO)CA or sequential HNCA experiments which provide unambiguous information of sequential correlations. However, this almost doubles the experimental time. Besides, they require repeated scanning through the (15)N planes to search for the matching peaks along the carbon dimension. In this background, we present here two pulse sequences, termed as hncoCANH and hnCOcaNH that lead to spectra equivalent to HNCA and HN(CA)CO spectra, respectively, but with direct distinction of inter- and intra-residue peaks; these occur with opposite signs in the new experiments. The two pulse sequences have been derived by simple modification of the previously described HN(C)N pulse sequence [Panchal et al., J. Biomol. NMR 20 (2001) 135-147] to frequency-label (13)C(alpha) or (13)C' instead of (15)N during the t(1) period. Like HN(C)N, these spectra also exhibit special patterns of self and sequential peaks around glycines and prolines, which enable direct identification of certain triplets of residues and thus provide internal checks during the sequential assignment walk. The spectra enable rapid and unambiguous assignment of H(N), (15)N and (13)C(alpha) (or (13)C') in a single experiment, and thus would be of great value in high-throughput structural proteomics.
在结构蛋白质组学研究中，节省数据采集时间是 NMR 脉冲序列开发的主要方向。常规用于序列残基归属的 HNCA 和 HN(CA)CO 脉冲序列的局限性在于它们不能区分残基间和残基内相关性。为了消除这种歧义，必须记录 HNCO 和 HN(CO)CA 或顺序 HNCA 实验，这些实验提供了序列相关性的明确信息。然而，这几乎将实验时间增加了一倍。此外，它们需要通过（15）N 平面重复扫描，以沿碳维搜索匹配的峰。在此背景下，我们提出了两种脉冲序列，分别称为 hncoCANH 和 hnCOcaNH，它们分别产生与 HNCA 和 HN(CA)CO 谱等效的谱，但具有残基间和残基内峰的直接区分；在新实验中，这些峰具有相反的符号。这两个脉冲序列是通过对先前描述的 HN(C)N 脉冲序列[Panchal 等人，J. Biomol. NMR 20（2001）135-147]进行简单修改得到的，即在 t(1)期间用（13）C(alpha)或（13）C'而不是（15）N 对（13）C(alpha)或（13）C'进行频率标记。与 HN(C)N 一样，这些谱也在甘氨酸和脯氨酸周围显示出特殊的自和序列峰模式，这可以直接识别某些残基的三联体，从而在序列归属过程中提供内部检查。这些谱可以在单个实验中快速且明确地分配 H(N)、（15）N 和（13）C(alpha)（或（13）C'），因此在高通量结构蛋白质组学中具有很高的价值。
