Riek R, Wider G, Pervushin K, Wüthrich K
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland.
Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):4918-23. doi: 10.1073/pnas.96.9.4918.
In common multidimensional NMR experiments for studies of biological macromolecules in solution, magnetization transfers via spin-spin couplings [insensitive nuclei enhanced by polarization transfer (INEPT)] are key elements of the pulse schemes. For molecular weights beyond 100,000, transverse relaxation during the transfer time may become a limiting factor. This paper presents a transfer technique for work with big molecules, cross relaxation-enhanced polarization transfer (CRINEPT), which largely reduces the size limitation of INEPT transfers with the use of cross-correlated relaxation-induced polarization transfer. The rate of polarization transfer by cross-correlated relaxation is proportional to the rotational correlation time, so that it becomes a highly efficient transfer mechanism for solution NMR with very high molecular weights. As a first implementation, [15N,1H]-correlation experiments were designed that make use of cross-correlation between dipole-dipole coupling and chemical shift anisotropy of the 15N---1H-moieties for both CRINEPT and transverse relaxation-optimized spectroscopy (TROSY). When compared with INEPT-based [15N,1H]-TROSY, these experiments yielded up to 3-fold signal enhancement for amide groups of a 110,000-Da protein in aqueous solution at 4 degrees C. CRINEPT opens avenues for solution NMR with supramolecular structures such as membrane proteins solubilized in micelles or lipid vesicles, proteins attached to nucleic acid fragments, or oligomeric proteins.
在用于研究溶液中生物大分子的常见多维核磁共振实验中，通过自旋 - 自旋耦合的磁化转移[通过极化转移增强的不灵敏核（INEPT）]是脉冲序列的关键要素。对于分子量超过100,000的情况，转移时间内的横向弛豫可能成为限制因素。本文提出了一种适用于大分子的转移技术——交叉弛豫增强极化转移（CRINEPT），它通过使用交叉相关弛豫诱导的极化转移，在很大程度上降低了INEPT转移的尺寸限制。交叉相关弛豫引起的极化转移速率与旋转相关时间成正比，因此对于非常高的分子量的溶液核磁共振来说，它成为一种高效的转移机制。作为首次应用，设计了[15N,1H]相关实验，该实验利用15N---1H基团的偶极 - 偶极耦合与化学位移各向异性之间的交叉相关性来进行CRINEPT和横向弛豫优化光谱（TROSY）。与基于INEPT的[15N,1H]-TROSY相比，这些实验在4摄氏度的水溶液中，对于110,000道尔顿蛋白质的酰胺基团产生了高达3倍的信号增强。CRINEPT为具有超分子结构的溶液核磁共振开辟了道路，例如溶解在胶束或脂质囊泡中的膜蛋白、附着于核酸片段的蛋白质或寡聚蛋白。
