Pervushin K, Riek R, Wider G, Wüthrich K
Institut für Molekularbiologie und Biophysik Eidgenössische Technische Hochschule Hönggerberg CH-8093 Zurich, Switzerland.
Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12366-71. doi: 10.1073/pnas.94.23.12366.
Fast transverse relaxation of 1H, 15N, and 13C by dipole-dipole coupling (DD) and chemical shift anisotropy (CSA) modulated by rotational molecular motions has a dominant impact on the size limit for biomacromolecular structures that can be studied by NMR spectroscopy in solution. Transverse relaxation-optimized spectroscopy (TROSY) is an approach for suppression of transverse relaxation in multidimensional NMR experiments, which is based on constructive use of interference between DD coupling and CSA. For example, a TROSY-type two-dimensional 1H,15N-correlation experiment with a uniformly 15N-labeled protein in a DNA complex of molecular mass 17 kDa at a 1H frequency of 750 MHz showed that 15N relaxation during 15N chemical shift evolution and 1HN relaxation during signal acquisition both are significantly reduced by mutual compensation of the DD and CSA interactions. The reduction of the linewidths when compared with a conventional two-dimensional 1H,15N-correlation experiment was 60% and 40%, respectively, and the residual linewidths were 5 Hz for 15N and 15 Hz for 1HN at 4 degrees C. Because the ratio of the DD and CSA relaxation rates is nearly independent of the molecular size, a similar percentagewise reduction of the overall transverse relaxation rates is expected for larger proteins. For a 15N-labeled protein of 150 kDa at 750 MHz and 20 degrees C one predicts residual linewidths of 10 Hz for 15N and 45 Hz for 1HN, and for the corresponding uniformly 15N,2H-labeled protein the residual linewidths are predicted to be smaller than 5 Hz and 15 Hz, respectively. The TROSY principle should benefit a variety of multidimensional solution NMR experiments, especially with future use of yet somewhat higher polarizing magnetic fields than are presently available, and thus largely eliminate one of the key factors that limit work with larger molecules.
1H、15N和13C通过偶极-偶极耦合（DD）以及由分子旋转运动调制的化学位移各向异性（CSA）引起的快速横向弛豫，对溶液中可通过核磁共振光谱研究的生物大分子结构的尺寸限制具有主要影响。横向弛豫优化光谱法（TROSY）是一种在多维核磁共振实验中抑制横向弛豫的方法，它基于对DD耦合和CSA之间干涉的建设性利用。例如，在750 MHz的1H频率下，对一个分子量为17 kDa的DNA复合物中均匀15N标记的蛋白质进行的TROSY型二维1H,15N相关实验表明，在15N化学位移演化过程中的15N弛豫以及信号采集过程中的1HN弛豫，都因DD和CSA相互作用的相互补偿而显著降低。与传统二维1H,15N相关实验相比，线宽的减小分别为60%和40%，在4℃时15N的剩余线宽为5 Hz，1HN的剩余线宽为15 Hz。由于DD和CSA弛豫率的比值几乎与分子大小无关，预计对于更大的蛋白质，整体横向弛豫率也会有类似的百分比降低。对于一个在750 MHz和20℃下的150 kDa的15N标记蛋白质，预计其15N的剩余线宽为10 Hz，1HN的剩余线宽为45 Hz，而对于相应的均匀15N,2H标记蛋白质，预计其剩余线宽分别小于5 Hz和15 Hz。TROSY原理应有利于各种多维溶液核磁共振实验，特别是在未来使用比目前可用的略高极化磁场的情况下，从而在很大程度上消除限制对更大分子进行研究的关键因素之一。
