Subtle Dynamics of holo Glutamine Binding Protein Revealed with a Rigid Paramagnetic Probe
Subtle Dynamics of holo Glutamine Binding Protein Revealed with a Rigid Paramagnetic Probe
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用刚性顺磁探针揭示全息谷氨酰胺结合蛋白的微妙动力学
DOI：
10.1021/bi4015715
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发表时间：
2014-03-11
期刊：
影响因子：
2.9
通讯作者：
Tang, Chun
中科院分区：
文献类型：
--
作者：
Liu, Zhu;Gong, Zhou;Tang, Chun
细菌周质结合蛋白(PBPs)参与周质内小分子的转运。要卸载，PBP的两个结构域打开，允许配体退出。然而，目前尚不清楚结合位点附近是否存在促进配体快速解离的动力学因素。为了可视化这种动力学，我们利用顺磁松弛增强(Pre)核磁共振，并将刚性顺磁探针引入到PBP，即与其同源配体结合的谷氨酰胺结合蛋白(QBP)。顺磁性的铜(II)离子被夹在螺旋上的工程双组氨酸基序和NTA封端分子之间。所提供的顺磁探头是如此坚硬，以至于从全息QBP的单一结构计算的预数值与观测值基本一致。然而，剩余的预差显示出与顺磁探头相反的区域中的环的动力学。根据Steed分子动力学模拟的评估，这个环与全息QBP中的谷氨酰胺配体包装在一起，并在配体解离时经历波动。因此，在纳秒到微秒的时间尺度内发生在小群体中的环动力学可能与配体解离过程有关。本文描述的刚性顺磁探针可以嫁接到其他蛋白质体系上进行结构和动力学研究。
Bacterial periplasmic binding proteins (PBPs) are involved in the translocation of small molecules in the periplasm. To unload, the two domains of a PBP open up, allowing the ligand to exit. However, it is not clear whether there are dynamics near the binding site which can facilitate the rapid dissociation of a ligand. To visualize such dynamics, we utilized paramagnetic relaxation enhancement (PRE) NMR and introduced a rigid paramagnetic probe to a PBP, glutamine-binding protein (QBP) with its cognate ligand bound. A paramagnetic Cu(II) ion is sandwiched between an engineered di-histidine motif at a helix and an NTA capping molecule. The afforded paramagnetic probe is so rigid that PRE values calculated from a single structure of holo QBP largely agree with the observed values. The remaining PRE discrepancies, however, manifest dynamics of a loop in the opposite domain from the paramagnetic probe. This loop packs against the glutamine ligand in the holo QBP and undergoes fluctuations upon ligand dissociation, as assessed by steered molecular dynamics simulations. As such, the loop dynamics, occurring for a small population in nanosecond to microsecond time scale, may be related to the ligand dissociation process. The rigid paramagnetic probe described herein can be grafted to other protein systems for structure and dynamics studies.