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

使用基于质谱的策略和计算分析对大型蛋白质复合物进行建模的综合方法。

• 批准号: 1622063
• 金额: --
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 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=studentship-1622063
• 关键词: integrative; approach; modelling; large; protein

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.

战略研究重点：工业生物技术和生物能源摘要这是一个新的结合质谱（MS）为基础的实验与蛋白质组装建模和生物信息学工具，用于研究瞬时蛋白质复合物的结构动力学。从MS生成的结构数据-天然MS，离子迁移率（IM）-MS和氢氘交换（HDX）-MS -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聚体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，即成簇的规则间隔回文重复序列（CRISPR）复合物。Project Cells包含物理相互作用蛋白质的大分子复合物。表征这些复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]，已成为研究其他难以处理的复合物结构的强大工具。发展和评估的混合动力复合物的表征方法形成了我们的多学科研究假设的基础。 该方法建立在基于MS的蛋白质复合物结构建模策略的基础上[2]。创新点是：a）HDX-MS实验的性能并将其纳入我们的混合方法中。这些实验报告了蛋白质不同区域内HDX速率的时间依赖性变化，从而获得有关其溶剂可及性的信息。HDX-MS测量结果将与根据MD模拟预测的蛋白质表面上的水的动力学行为计算的溶剂密度图相证实[3]; B）当单个亚基组装成功能复合物时对其动态重排的处理。我们将MS引导的Monte Carlo采样与溶剂MD模拟交叉，使我们能够监测与HDX-MS相当的时间尺度上组装体的构象变化。最后，蛋白质-蛋白质相互作用网络分析[4]将使我们能够注释和推断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，我们将评估其在不同复合体的基准中再现近原生模型的能力。接下来，我们将靶向CRISPR，这是一种基于RNA的免疫系统，负责消除遗传寄生虫，用于MS实验的纯化样品可从我们的合作者（Malcolm白色，圣安德鲁斯）获得。以前，我们使用生物化学方法，电子显微镜，MS和建模对CRISPR-CSM复合物进行建模[5]。在这里，我们将靶向由七种蛋白质组成的硫磺硫化叶菌CRISPR-CMR。CMR的详细结构和功能表征将揭示其在靶向病毒核酸中的作用。

项目成果

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry.

• DOI: 10.3791/57966
• 发表时间: 2018-10-15
• 期刊: Journal of visualized experiments : JoVE
• 作者: Ahdash Z;Lau AM;Martens C;Politis A
• 通讯作者: Politis A

Surface Accessibility and Dynamics of Macromolecular Assemblies Probed by Covalent Labeling Mass Spectrometry and Integrative Modeling.

• DOI: 10.1021/acs.analchem.6b02875
• 发表时间: 2017-02-07
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: Schmidt C;Macpherson JA;Lau AM;Tan KW;Fraternali F;Politis A
• 通讯作者: Politis A

A Mass-Spectrometry-Based Modelling Workflow for Accurate Prediction of IgG Antibody Conformations in the Gas Phase.

基于质谱的建模工作流程，用于准确预测气相中IgG抗体构象。

• DOI: 10.1002/anie.201812018
• 发表时间: 2018-12-21
• 期刊: Angewandte Chemie (International ed. in English)
• 作者: Hansen K;Lau AM;Giles K;McDonnell JM;Struwe WB;Sutton BJ;Politis A
• 通讯作者: Politis A

Mechanistic insight into the assembly of the HerA-NurA helicase-nuclease DNA end resection complex.

• DOI: 10.1093/nar/gkx890
• 发表时间: 2017-11-16
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Ahdash Z;Lau AM;Byrne RT;Lammens K;Stüetzer A;Urlaub H;Booth PJ;Reading E;Hopfner KP;Politis A
• 通讯作者: Politis A

Direct protein-lipid interactions shape the conformational landscape of secondary transporters.

• DOI: 10.1038/s41467-018-06704-1
• 发表时间: 2018-10-08
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Martens C;Shekhar M;Borysik AJ;Lau AM;Reading E;Tajkhorshid E;Booth PJ;Politis A
• 通讯作者: Politis A