Department of Structural Biology, St Jude Children's Research Hospital, Memphis, TN, 38105, USA.
Departments of Molecular Genetics, Biochemistry and Chemistry, University of Toronto, Toronto, ON, M5S 1A8, Canada.
J Biomol NMR. 2020 May;74(4-5):213-221. doi: 10.1007/s10858-020-00310-4. Epub 2020 Apr 2.
The 3D HCCH-TOCSY and HCC(CO)NH-TOCSY experiments provide through bond connectivity and are used for side-chain chemical shift assignment by solution-state NMR. Careful design and implementation of the pulse sequence are key to the successful application of the technique particularly when trying to extract the maximum information out of challenging biomolecules. Here we investigate the source of and propose solutions for abnormal peak splitting ranging from 152 to 80 Hz and below that were found in three popular TOCSY-based experiment types: H(F)-C(F)-DIPSI-H(F), C(F)-DIPSI-C(F)-H(F), and C(F)-DIPSI-N(F)-H(F). Peak splitting occurs in the indirect C(F) or C(F) dimension before DIPSI and analyses indicate that the artifacts are resulted mainly from the DIPSI transforming a double spin order [Formula: see text] from C-C scalar J coupling during t into observable megnetization. The splitting is recapitulated by numerical simulation and approaches are proposed to remove it. Adding a pure delay of 3.7 ms immediately before DIPSI is a simple and effective strategy to achieve 3D HCCH-TOCSY and HCC(CO)NH-TOCSY spectra free of splitting with full crosspeak intensity.
3D HCCH-TOCSY 和 HCC(CO)NH-TOCSY 实验提供了键连接，并通过溶液 NMR 用于侧链化学位移分配。脉冲序列的精心设计和实施是该技术成功应用的关键，特别是在试图从具有挑战性的生物分子中提取最大信息量时。在这里，我们研究了在三种流行的基于 TOCSY 的实验类型中发现的异常峰分裂的来源，并提出了解决方案，峰分裂范围从 152 到 80 Hz 及以下：H(F)-C(F)-DIPSI-H(F)、C(F)-DIPSI-C(F)-H(F) 和 C(F)-DIPSI-N(F)-H(F)。在 DIPSI 之前，间接的 C(F)或 C(F)维度中出现峰分裂，分析表明，这些伪影主要是由于 DIPSI 在 t 期间将双自旋顺序[公式：见正文]从 C-C 标量 J 耦合转换为可观察的磁化。通过数值模拟再现了分裂，并提出了去除分裂的方法。在 DIPSI 之前立即添加 3.7 ms 的纯延迟是一种简单而有效的策略，可以实现无分裂的 3D HCCH-TOCSY 和 HCC(CO)NH-TOCSY 谱，具有全交叉峰强度。
