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）光谱是一种用于结构生物学领域应用的新兴技术。具体而言，使用称为Peldor的EPR方法（PULSETERON-ELECTRON双共振）或鹿（双电子电子共振），可以可靠地测量选择系统中两个顺磁心中心之间的纳米范围内的距离。这些顺磁中心可以是本地金属离子或自由基辅助因子，但最常见的是，它们是通过一种称为位置定向自旋标签的技术故意引入的。近年来，Peldor技术已越来越多地应用于复杂的生物系统，包括（同型）低聚物，即同一成分的几个副本。即使每个组成部分仅连接一个自旋标签，这也导致了多个自旋中心的存在。这些多旋转系统比已建立的两型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，将结果混淆很高。但是，最近提出了几种缓解这些局限性的方法（修改实验或分析）。我们提出的研究旨在获取结构信息，以补充EPR在多聚体复合物上的既定方法。在这里，我们希望将单个成分内的距离与复合物成分之间的所有可能的距离区分开。换句话说，虽然已建立的方法基于测量单体中的距离距离形成了多聚体并带有一个自旋标签，但我们希望靶向多组分的一个双标记的单体中的一个双标记的单体。保持样品的所有其他方面制备的所有其他方面都会制备相同的同样，这一双重标记将两倍的型号与Spin-Labels融合在一起的两倍。这将严重增加由多旋转效应引起的问题，并另外将复杂性增加了更多的不同自旋间距离。此外，在标准方法中，将不可能区分现有的距离内感兴趣的距离与存在的其他距离。因此，我们提出了一项原则研究，以解决对知识内距离必须可靠提取的偏好。换句话说，必须用未标记的单体“稀释”双重标记的单体？这将以整体方法来解决，以整合数值模拟，合成模型系统以及用于数据采集和处理的新方法，以证明对生物样品的适用性。此外，我们将获得所获得的知识转移到模仿生物系统中不同二聚体平衡的更复杂的模型系统。在该项目的最后阶段，提取的原理应应用于合适的多聚体蛋白质，以证明结构生物学的新方法的价值。这项研究将显着提高EPR领域的当前知识和方法，尤其是在Peldor上对Peldor on蛋白质上的知识和方法。此外，我们的方法将增加结构技术的军械库，并可能允许在特定系统中应对结构性挑战，而这些系统迄今无法使用的方法。