Fatty Acid Triangulation in Albumins Using a Landmark Spin Label
Fatty Acid Triangulation in Albumins Using a Landmark Spin Label
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DOI：
10.1002/ijch.201900073
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发表时间：
2019-11-18
影响因子：
3.2
通讯作者：
Hinderberger, Dariush
中科院分区：
文献类型：
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作者：
Reichenwallner, Joerg;Haucnschild, Till;Hinderberger, Dariush
用双电子-电子共振(DER)技术发现了蛋白中多达6个脂肪酸(FA)结合部位的多个空间相关性。在电子顺磁共振(EPR)谱中使用了结合自旋标记和自旋探测技术的策略。这是通过在EPR活性的顺磁性硬脂酸衍生物和白蛋白的自组装系统中引入额外的共价标志性自旋(LS)标记来实现的。因此，半胱氨酸特异的顺磁性LS被固定在白蛋白表面的唯一位置(Cys34)，为监测统计配体摄取提供了固定的拓扑参考点。我们认为，确定LS和EPR活性脂肪酸衍生物之间出现的纳米级距离分布通常允许直接观察溶液中单独占据的结合位点。从本质上讲，可以从这种FA-LS自旋间关联中追踪到几个结合口袋、几组结合口袋以及配体诱导变构调节的证据。实验结果得到了来自分子动力学(MD)模拟的理论预测的证实。观察到，即使在最低的配体负载量水平上，白蛋白系综中的所有结合部位也可以在统计上被填充。这种方法通常具有使用这种自旋标记的配体来定位蛋白质中单个配体结合位置的占据状态的潜力。
Several spatial correlations of up to six fatty acid (FA) binding sites in albumins were found by double electron-electron resonance (DEER). A strategy was used that combines spin-labeling and spin-probing techniques in electron paramagnetic resonance (EPR) spectroscopy. This is here achieved by introducing an additional covalent landmark spin (LS) label to the self-assembled system of EPR-active, paramagnetic stearic acid derivatives and albumins. Therefore, a cysteine specific, paramagnetic LS that was attached to the albumin surface at a unique position (Cys34) provides a fixed topological reference point for monitoring statistical ligand uptake. We propose that the determination of nanoscale distance distributions emerging between the LS and EPR-active fatty acid derivatives generally allows for the direct observation of individually occupied binding sites in solution. Essentially, several binding pockets, groups of them and evidence for ligand-induced allosteric modulation can be traced from such FA-LS interspin correlations. Experimental results were substantiated with theoretical predictions from molecular dynamics (MD) simulations. It was observed that all binding sites in an albumin ensemble may be statistically filled even at the lowest level of ligand loading. This approach generally bears the potential for mapping occupation states of individual ligand binding sites in proteins using such spin-labeled ligands.