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Conformational flexibility of adenine riboswitch aptamer in apo and bound states using NMR and an X-ray free electron laser.
Journal of Biomolecular NMR ( IF 2.4 ) Pub Date : 2019-10-14 , DOI: 10.1007/s10858-019-00278-w
Jienv Ding 1 , Monalisa Swain 1 , Ping Yu 1 , Jason R Stagno 1 , Yun-Xing Wang 1
Affiliation  

Riboswitches are structured cis-regulators mainly found in the untranslated regions of messenger RNA. The aptamer domain of a riboswitch serves as a sensor for its ligand, the binding of which triggers conformational changes that regulate the behavior of its expression platform. As a model system for understanding riboswitch structures and functions, the add adenine riboswitch has been studied extensively. However, there is a need for further investigation of the conformational dynamics of the aptamer in light of the recent real-time crystallographic study at room temperature (RT) using an X-ray free electron laser (XFEL) and femtosecond X-ray crystallography (SFX). Herein, we investigate the conformational motions of the add adenine riboswitch aptamer domain, in the presence or absence of adenine, using nuclear magnetic resonance relaxation measurements and analysis of RT atomic displacement factors (B-factors). In the absence of ligand, the P1 duplex undergoes a fast exchange where the overall molecule exhibits a motion at kex ~ 319 s-1, based on imino signals. In the presence of ligand, the P1 duplex adopts a highly ordered conformation, with kex~ 83 s-1, similar to the global motion of the molecule, excluding the loops and binding pocket, at 84 s-1. The µs-ms motions in both the apo and bound states are consistent with RT B-factors. Reduced spatial atomic fluctuation, ~ 50%, in P1 upon ligand binding coincides with significantly attenuated temporal dynamic exchanges. The binding pocket is structured in the absence or presence of ligand, as evidenced by relatively low and similar RT B-factors. Therefore, despite the dramatic rearrangement of the binding pocket, those residues exhibit similar spatial thermal fluctuation before and after binding.

中文翻译:

使用NMR和X射线自由电子激光,在apo和键合状态的腺嘌呤核糖开关适体的构象柔性。

核糖开关是主要在信使RNA的非翻译区中发现的结构顺式调节子。核糖开关的适体结构域用作其配体的传感器,其配体的结合触发构象改变,该构象改变调节其表达平台的行为。作为用于理解核糖开关的结构和功能的模型系统,已对添加腺嘌呤核糖开关进行了广泛的研究。然而,根据最近使用X射线自由电子激光(XFEL)和飞秒X射线晶体学在室温(RT)上进行的实时晶体学研究,需要进一步研究适体的构象动力学( SFX)。在这里,我们研究在存在或不存在腺嘌呤的情况下,添加腺嘌呤核糖开关适体结构域的构象运动,使用核磁共振弛豫测量和RT原子位移因子(B因子)分析。在没有配体的情况下,P1双链体经历快速交换,其中基于亚氨基信号,整个分子在kex〜319 s-1处显示运动。在配体存在的情况下,P1双链体具有高度有序的构象,其kex_ 83 s-1与分子的整体运动相似,但不包括环和结合口袋,为84 s-1。载脂蛋白和结合态的μs-ms运动均与RT B因子一致。配体结合后,P1中的空间原子波动降低了〜50%,这与时间动态交换显着减弱相吻合。结合口袋的结构是在不存在或存在配体的情况下进行的,这是由相对较低且相似的RT B因子所证实的。因此,
更新日期:2019-10-12
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