Electron Paramagnetic Resonance - Volume 27
Electron Paramagnetic Resonance - Volume 27
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电子顺磁共振 - 第 27 卷
DOI：
10.1039/9781839162534-00074
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Hartley A
中科院分区：
文献类型：
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作者：
Hartley A
脉冲EPR方法，如PELDOR（或DEER）和ESEEM已成为评估膜蛋白构象，折叠，寡聚化和动力学的强大工具。PELDOR距离测量可以提供关于膜蛋白的构象平衡的定量信息，与诸如X射线晶体学和cryo-EM的技术很好地结合。ESEEM光谱可以在单个残留物水平上提供氘（或溶剂）可及性量化。对膜蛋白研究的固有限制是需要将蛋白从其天然膜环境中除去。然而，膜蛋白领域的最新发展提供了模拟该环境的脂质支架，以限制蛋白质溶解的影响并提供脂质组合物选择的灵活性，这是膜蛋白功能完整性的关键因素。此外，计算工具可以用来帮助自旋标记实验，或定制纳入实验推导PELDOR距离的限制，以揭示完整的构象合奏膜蛋白。在本章中，我们将重点介绍脉冲EPR在膜蛋白研究中的应用，重点介绍案例研究，包括我们在机械敏感离子通道方面的工作。我们将讨论实验的考虑，并介绍一些计算工具，可以结合脉冲EPR方法。
Pulsed EPR methods, such as PELDOR (or DEER) and ESEEM have emerged as powerful tools for assessing membrane protein conformation, folding, oligomerisation and dynamics. PELDOR distance measurements can provide quantitative information on the conformational equilibria of membrane proteins, dovetailing well with techniques such as X-ray crystallography and cryo-EM. ESEEM spectroscopy can offer deuterium (or solvent) accessibility quantification at a single residue level. An inherent limit placed on membrane protein studies is the need to remove the protein from its natural membrane environment. However, recent developments in the membrane protein field provide lipid scaffolds that mimic that environment, to limit the impact of protein solubilisation and offer flexibility of choice in lipid composition, a key factor in the functional integrity of membrane proteins. Additionally, computational tools can be used to aid spin-labelling experiments, or tailored to incorporate experimentally derived PELDOR distance restraints to reveal the complete conformational ensemble of membrane proteins. In this chapter, we will focus on the application of pulsed EPR in the study of membrane proteins, highlighting case studies including our work on mechanosensitive ion channels. We will discuss experimental considerations, and introduce some of the computational tools that can be used in combination with pulsed EPR methods.