An integrative approach for modelling large protein complexes using mass spectrometry-based strategies and computational analyses.

摘要本项目旨在将基于质谱（MS）的实验与蛋白质组装建模和生物信息学工具相结合，研究瞬态蛋白质复合物的结构动力学。从MS（原生MS）、离子迁移率(IM)-MS和氢氘交换(HDX)-MS -生成的结构数据将被编码到建模约束中，随后通过蛋白质-蛋白质相互作用网络进行分析，以构建3D蛋白质组装模型。我们将在具有已知晶体结构的异质复合物的基准上验证我们的方法，并使用它来表征最近发现的抗病毒防御系统的组装动力学，聚集规则间隔回文重复（CRISPR）复合物。项目细胞包含物理上相互作用的蛋白质的大分子复合物。表征这些配合物的结构动力学仍然是一个挑战，使用单一的方法。混合方法集成了多种类型的生物物理数据[1]，已经成为研究其他难以处理的复合物结构的强大工具。开发和评估表征动态复合物的混合方法构成了我们多学科研究假设的基础。该方法建立在突破性的基于质谱的蛋白质复合物[2]结构建模策略之上。创新之处在于：a)将HDX-MS实验的性能和结合到我们的混合方法中。这些实验报告了蛋白质不同区域内HDX率的时间依赖性变化，从而获得了有关其溶剂可及性的信息。HDX-MS测量结果将与MD模拟预测的水在蛋白质表面的动力学行为计算的溶剂密度图相证实；B)处理单个亚基在组装成功能复合物时的动态重排。我们将交叉ms引导的蒙特卡罗采样与溶剂MD模拟，使我们能够监测与HDX-MS相当的时间尺度上组件的构象变化。最后，蛋白质-蛋白质相互作用网络分析[4]将允许我们注释和推断我们实验中观察到的生物复合物功能的置信度。在将我们的方法应用于未知的生物复合体之前，我们将评估其在多种复合体的基准中再现近原生模型的能力。接下来，我们将瞄准CRISPR，这是一种基于rna的免疫系统，负责消除遗传寄生虫，我们的合作者（Malcolm White, St. Andrews）提供了用于MS实验的纯化样品。在此之前，我们使用生化方法、电子显微镜、质谱和建模[5]对CRISPR-CSM复合体进行了建模。在这里，我们将针对由7个蛋白组成的solfataricus的CRISPR-CMR。CMR的详细结构和功能特征将揭示其靶向病毒核酸的作用。

Strategic Research Priority: Industrial biotechnology and bioenergyAbstract This is to integrate a novel combination of mass spectrometry (MS)-based experiments with protein assembly modelling and bioinformatics tools for studying the structural dynamics of transient protein complexes. Structural data generated from MS - native MS, ion mobility (IM)-MS and hydrogen deuterium exchange (HDX)-MS - will be encoded into modelling restraints and subsequently analysed by protein-protein interaction networks for building 3D protein assembly models. We will validate our method on a benchmark of heteromeric complexes with known crystal structures and use it to characterize the assembly dynamics of the recently discovered antiviral defense system, clustered regularly interspaced palindromic repeats (CRISPR) complex. Project Cells contain macromolecular complexes of physically interacting proteins. Characterizing the structural dynamics of these complexes remains a challenge using single methods. Hybrid methods, which integrate multiple types of biophysical data[1], have emerged as a powerful tool for studying the architectures of otherwise intractable complexes. The development and assessment of a hybrid method for characterizing dynamic complexes forms the basis of our multi-disciplinary research hypothesis. The method builds upon a ground-breaking MS-based strategy for structural modelling of protein complexes[2]. The innovation is: a) the performance and incorporation of HDX-MS experiments into our hybrid method. These experiments report time-dependent changes of HDX rates within different regions of proteins, yielding information about their solvent accessibility. HDX-MS measurements will be corroborated with calculated solvent density maps from dynamical behavior of water on protein surface predicted by MD simulations[3]; b) the treatment of dynamic rearrangements of the individual subunits as they assemble into functional complexes. We will intersect an MS-guided Monte Carlo sampling with solvent MD simulations enabling us to monitor the conformational changes of assemblies over time scales comparable to HDX-MS. Finally, protein-protein interaction network analyses[4] will allow us to annotate and infer confidence to the function of biological complexes observed in our experiments. Prior to applying our method in unknown biological complexes, we will assess its ability in reproducing near-native models in a benchmark of diverse complexes. Next, we will target the CRISPR, an RNA-based immune system responsible for eliminating genetic parasites, for which purified samples for MS experiments are available from our collaborator (Malcolm White, St. Andrews). Previously, we modelled the CRISPR-CSM complex using biochemical methods, electron microscopy, MS and modelling[5]. Here we will target the CRISPR-CMR from Sulfolobus solfataricus composed of seven proteins. The detailed structural and functional characterisation of CMR will reveal its role in targeting viral nucleic acids.