Intra-monomer EPR distances in multimeric systems

电子顺磁共振波谱是一种新兴的结构生物学技术。具体来说，使用一种称为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因子，但最常见的是，它们是通过一种称为定点自旋标记的技术故意引入的。近年来，PELDOR技术越来越多地应用于复杂的生物系统，由（homo-）寡聚物组成，即相同成分的几个拷贝。这导致了多个自旋中心的存在，即使每个成分只附着一个自旋标签。这些多自旋系统远比已建立的双自旋系统更具挑战性。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，在更复杂的病例中，混淆结果的可能性很大。然而，最近提出了几种方法（修改实验或分析）来缓解这些局限性。我们提出的研究目的是获取结构信息，以补充现有的多聚体配合物的EPR方法。在这里，我们想要区分单个成分内部的距离和所有可能的复合物成分之间的距离。换句话说，虽然所建立的方法是基于测量形成多聚体的单体之间的距离，并且每个单体具有一个自旋标记，但我们想要测量多聚体的一个双标记单体内的距离。保持样品制备的所有其他方面相同，这种双重标签将使旋转标签的数量增加一倍。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现有的其他距离区分开来。因此，我们提出了一个原理证明研究，解决了多少优先考虑要可靠地提取单体内距离。换句话说，有双重标签的单体需要用没有标签的单体“稀释”多少？这将通过综合数值模拟、综合模型系统和数据采集和处理的新方法来解决，以证明在生物样品上的适用性。此外，我们将把获得的知识转移到更复杂的模型系统，模拟生物系统中不同的二聚化平衡。在这个项目的最后阶段，提取的原理将应用于一个合适的多聚体蛋白质，以证明结构生物学新方法的价值。这项研究将显著推进EPR领域的现有知识和方法，特别是关于蛋白质的PELDOR。此外，我们的方法将增加结构技术的武器库，并可能允许解决特定系统中的结构挑战，这些挑战是迄今为止可用的方法无法实现的。

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.