NMR Laboratory, Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory, University of Helsinki, P.O. Box 65, 00014, Helsinki, Finland.
J Biomol NMR. 2010 Jul;47(3):183-94. doi: 10.1007/s10858-010-9422-z. Epub 2010 May 8.
We describe a novel pulse sequence, MQ-HNCO-TROSY, for the measurement of scalar and residual dipolar couplings between amide proton and nitrogen in larger proteins. The experiment utilizes the whole 2T(N) polarization transfer delay for labeling of (15)N chemical shift in a constant time manner, which efficiently doubles the attainable resolution in (15)N dimension with respect to the conventional HNCO-TROSY experiment. In addition, the accordion principle is employed for measuring (J + D)(NH)s, and the multiplet components are selected with the generalized version of the TROSY scheme introduced by Nietlispach (J Biomol NMR 31:161-166, 2005). Therefore, cross peak overlap is diminished while the time period during which the (15)N spin is susceptible to fast transverse relaxation associated with the anti-TROSY transition is minimized per attainable resolution unit. The proposed MQ-HNCO-TROSY scheme was employed for measuring RDCs in high molecular weight protein IgFLNa16-21 of 557 residues, resulting in 431 experimental RDCs. Correlations between experimental and back-calculated RDCs in individual domains gave relatively low Q-factors (0.19-0.39), indicative of sufficient accuracy that can be obtained with the proposed MQ-HNCO-TROSY experiment in high molecular weight proteins.
我们描述了一种新的脉冲序列 MQ-HNCO-TROSY，用于测量酰胺质子和氮之间的标量和剩余偶极耦合在较大的蛋白质。该实验利用整个 2T（N）极化转移延迟以恒定时间的方式标记（15）N 化学位移，与传统的 HNCO-TROSY 实验相比，这有效地将（15）N 维度上可达到的分辨率提高了一倍。此外，采用手风琴原理测量（J + D）（NH）s，并用 Nietlispach 引入的广义 TROSY 方案（J Biomol NMR 31：161-166，2005）选择多峰分量。因此，交叉峰重叠减少，而（15）N 自旋易受与反 TROSY 跃迁相关的快速横向弛豫影响的时间周期在每个可达到的分辨率单元中最小化。所提出的 MQ-HNCO-TROSY 方案用于测量 557 个残基的高分子量蛋白质 IgFLNa16-21 的 RDC，得到 431 个实验 RDC。在各个域中实验 RDC 和反向计算 RDC 之间的相关性给出了相对较低的 Q 因子（0.19-0.39），表明在高分子量蛋白质中，所提出的 MQ-HNCO-TROSY 实验可以获得足够的准确性。
