Lee W, Revington M, Farrow N A, Nakamura A, Utsunomiya-Tate N, Miyake Y, Kainosho M, Arrowsmith C H
Division of Molecular and Structural Biology, Ontario Cancer Institute, University of Toronto, Canada.
J Biomol NMR. 1995 Jun;5(4):367-75. doi: 10.1007/BF00182280.
[ul-13C/15N]-L-tryptophan was prepared biosynthetically and its dynamic properties and intermolecular interaction with a complex of Escherichia coli trp-repressor and a 20 base-pair operator DNA were studied by heteronuclear isotope-edited NMR experiments. The resonances of the free and bound corepressor (L-Trp) were unambiguously identified from gradient-enhanced 15N-1H HSQC, 13C-1H HSQC, 13C- and 15N-edited 2D NOESY spectra. The exchange off-rate of the corepressor between the bound and free states was determined to be 3.4 +/- 0.52 s-1 at 45 degrees C, almost three orders of magnitude faster than the dissociation of the protein-DNA complex. Examination of the experimental NOE buildup curves indicates that it may be desirable to use longer mixing times than would normally be used for a large molecule, in order to detect weak intermolecular NOEs in the presence of exchange. Intermolecular NOEs from bound corepressor to trp-repressor and DNA were analyzed with respect to the mechanism of ligand exchange. This analysis suggests that, in order for the ligand to diffuse out of the complex, there must be significant movement or 'breathing' of the protein and/or DNA.
通过生物合成制备了[ul-13C/15N]-L-色氨酸，并利用异核同位素编辑核磁共振实验研究了其动态特性以及与大肠杆菌色氨酸阻遏物和一个20碱基对操纵子DNA复合物的分子间相互作用。从梯度增强的15N-1H HSQC、13C-1H HSQC、13C和15N编辑的二维NOESY谱中明确鉴定了游离和结合的辅阻遏物（L-色氨酸）的共振信号。在45℃下，辅阻遏物在结合态和游离态之间的交换解离速率被确定为3.4±0.52 s-1，几乎比蛋白质-DNA复合物的解离快三个数量级。对实验性NOE积累曲线的研究表明，为了在存在交换的情况下检测到弱的分子间NOE，可能需要使用比通常用于大分子的混合时间更长的时间。关于配体交换机制，分析了从结合的辅阻遏物到色氨酸阻遏物和DNA的分子间NOE。该分析表明，为了使配体从复合物中扩散出来，蛋白质和/或DNA必须有显著的移动或“呼吸”。
