MQ-HNCO-TROSY for the measurement of scalar and residual dipolar couplings in larger proteins: application to a 557-residue IgFLNa16-21
MQ-HNCO-TROSY for the measurement of scalar and residual dipolar couplings in larger proteins: application to a 557-residue IgFLNa16-21
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DOI：
10.1007/s10858-010-9422-z
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发表时间：
2010-07-01
影响因子：
2.7
通讯作者：
Permi, Perttu
中科院分区：
文献类型：
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作者：
Mantylahti, Sampo;Koskela, Outi;Permi, Perttu
我们描述了一种新的脉冲序列，MQ-HNCO-TROSY，用于测量较大蛋白质中酰胺质子与氮之间的标量偶极耦合和剩余偶极耦合。该实验利用整个2T(N)偏振传递延迟以恒定时间的方式标记N-15化学位移，与传统的HNCO-TROSY实验相比，有效地将N-15维的分辨率提高了一倍。此外，使用手风琴原理测量(J+D)(NH)S，并使用Nietlispach提出的推广版本的TROSY方案(J Biool NMR31：161-166,2005)来选择多重分量。因此，对于每个可达到的分辨率单位，交叉峰重叠被减小，同时N-15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被最小化。MQ-HNCO-TROSY方法被用来测量557个残基中的高分子量蛋白质IgFLNa16-21中的RDC，得到了431个实验RDC。实验和反向计算的各个结构域的RDC之间的关联给出了相对较低的Q因子(0.19-0.39)，表明通过所提出的MQ-HNCO-TROSY实验可以获得足够的精度，用于高分子量蛋白质。
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 N-15 chemical shift in a constant time manner, which efficiently doubles the attainable resolution in N-15 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 N-15 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.