Intra-monomer EPR distances in multimeric systems

多聚体系中单体内 EPR 距离

基本信息

批准号：
EP/M024660/1
负责人：
Bela Bode
金额：
$ 12.55万
依托单位：
University of St Andrews
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2015
资助国家：
英国
起止时间：
2015 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=EP%2FM024660%2F1
关键词：
Intra monomer EPR distances multimeric

项目摘要

Electron paramagnetic resonance (EPR) spectroscopy is an emerging technique for applications in the field of structural biology. Specifically, using an EPR method called PELDOR (pulsed electron-electron double resonance) or DEER (double electron-electron resonance), it is possible to reliably measure distances in the nanometre range between two paramagnetic centres in the system of choice. These paramagnetic centres can be native metal ions or radical cofactors, but most commonly they are deliberately introduced by a technique called site-directed spin-labelling. In recent years, the PELDOR technique has been increasingly applied to complex biological systems, consisting of (homo-)oligomers, i.e. several copies of the same constituent. This leads to the presence of multiple spin centres even though only one spin-label per constituent is attached. These multi-spin systems are by far more challenging than the established two-spin systems. The theory behind the methods used to extract the distance information from the experimental data is limited to two spin systems. Thus, there is a high potential to confound the results in the more complicated cases. However, several approaches (modifying experiment or analysis) to relieve these limitations have been suggested recently.With the research we propose we aim at obtaining structural information which is complementing the established methods for EPR on multimeric complexes. Here, we want to distinguish the distance within a single constituent from all those possible in-between the constituents of a complex. In other words, while the established approach is based on measuring the distance in-between monomers forming the multimers and bearing one spin-label each, we want to target distances within one doubly-labelled monomer of the multimer.Keeping all other aspects of the sample preparation the same, this double labelling will double the number of spin-labels incorporated. This will severely increase the issues caused by multi-spin effects and additionally increase complexity by a higher number of different inter-spin distances. Furthermore, in the standard approach it will be impossible to distinguish the intra-monomer distance of interest from the other distances present. Thus, we propose a proof-of-principle study addressing how much preference has to be given to the intra-monomer distance to be reliably extracted. In other words, how much does the doubly-labelled monomer have to be "diluted" with un-labelled monomer? This will be addressed in a holistic approach integrating numerical simulations, synthetic model systems and new approaches for data acquisition and processing to demonstrate applicability on biological samples. In addition, we will transfer the gained knowledge to more complex model systems mimicking different dimerisation equilibria in biological systems. In the final stage of this project, the extracted principles are to be applied to a suitable multimeric protein to demonstrate the value of the new approach for structural biology.This study will significantly advance the current knowledge and methodology in the field of EPR, especially with respect to PELDOR on proteins. Furthermore, our approach will add to the armoury of structural techniques and may allow tackling structural challenges in specific systems which are not accessible with the methods available to date.

电子顺磁共振（EPR）光谱是一种应用于结构生物学领域的新兴技术。具体来说，使用称为 PELDOR（脉冲电子-电子双共振）或 DEER（双电子-电子共振）的 EPR 方法，可以可靠地测量所选系统中两个顺磁中心之间的纳米范围内的距离。这些顺磁中心可以是天然金属离子或自由基辅助因子，但最常见的是它们是通过称为定点自旋标记的技术有意引入的。近年来，PELDOR 技术越来越多地应用于由（同）低聚物（即同一成分的多个副本）组成的复杂生物系统。这导致存在多个自旋中心，即使每个成分仅附着一个自旋标签。这些多旋转系统比现有的双旋转系统更具挑战性。用于从实验数据中提取距离信息的方法背后的理论仅限于两个自旋系统。因此，在更复杂的情况下很可能会混淆结果。然而，最近提出了几种缓解这些限制的方法（修改实验或分析）。通过我们的研究，我们的目标是获得结构信息，以补充多聚体复合物的 EPR 既定方法。在这里，我们想要区分单个成分内的距离与复合体成分之间所有可能的距离。换句话说，虽然所建立的方法是基于测量形成多聚体并各自带有一个自旋标签的单体之间的距离，但我们希望目标是多聚体的一个双标记单体内的距离。样品制备相同，这种双重标记将使掺入的自旋标记数量加倍。这将严重增加由多自旋效应引起的问题，并且由于更多不同的自旋间距离而额外增加复杂性。此外，在标准方法中，不可能将感兴趣的单体内距离与存在的其他距离区分开。因此，我们提出了一项原理验证研究，解决必须优先考虑单体内距离才能可靠提取的问题。换句话说，双标记单体必须用未标记单体“稀释”多少？这将通过集成数值模拟、合成模型系统和数据采集和处理新方法的整体方法来解决，以证明其对生物样本的适用性。此外，我们将把获得的知识转移到更复杂的模型系统中，模拟生物系统中不同的二聚平衡。在该项目的最后阶段，提取的原理将应用于合适的多聚蛋白，以证明结构生物学新方法的价值。这项研究将显着推进 EPR 领域的当前知识和方法，特别是尊重蛋白质上的 PELDOR。此外，我们的方法将增加结构技术的库，并可能允许解决特定系统中迄今为止可用的方法无法解决的结构挑战。