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Development of global analysis tools to model protein isotope exchange behaviour

开发用于模拟蛋白质同位素交换行为的全局分析工具

基本信息

批准号：
1862995
负责人：
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：
2016
资助国家：
英国
起止时间：
2016 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=studentship-1862995
关键词：
Development global analysis tools model

项目摘要

Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) is a rapid and sensitive technique to characterise protein structure, dynamics and interfaces. The overarching goal of this project is the development of global analysis tools to characterise protein HDX patterns to understand protein structure and assembly. Preliminary outputs indicate that there is scope for improvement through extensive benchmarking and optimisation. This will require the overexpression and purification of a range of carefully selected proteins that form binary interactions. The proteins must have known structures in their bound and unbound states and no significant structural changes must take place on assembly formation. There are a range of suitable candidates such as the Mms2-Ubc13 binary complex for which expression plasmids can be sourced commercially from the Addgene repository. Experimental HDX-MS difference plots will be acquired for each protein and the RMSE between these outputs and simulated profiles for the native assemblies used to guide the optimisation procedure. Subsequent to the development and optimisation of the approach it will be applied to understand the organisation between the Staphylococcal repressor protein (Stl) and the 11 dUTPase. The dUTPase-Stl interaction has been extensively characterised with a range of biophysical methods but the structure of the dUTPase-Stl assembly has not yet been successfully crystallised. A range of potential complexes has been generated by molecular docking but there is no way to critically evaluate the different docking poses. In a collaborative effort, we shall acquire experimental HDX-MS data for the dUTPase-Stl complex and then apply our procedure to to simulate HDX-MS difference outputs for the candidate assemblies and evaluate them on the basis of their agreement with experimental outputs.

氢氘交换质谱（HDX-MS）是一种快速、灵敏的蛋白质结构、动力学和界面分析技术。该项目的总体目标是开发全球分析工具，以分析蛋白质HDX模式，从而了解蛋白质结构和组装。初步产出表明，通过广泛的基准测试和优化，仍有改进的余地。这将需要过表达和纯化一系列精心选择的蛋白质，形成二元相互作用。蛋白质在其结合和未结合状态下必须具有已知的结构，并且在组装形成时必须不发生显著的结构变化。存在一系列合适的候选物，例如Mms 2-Ubc 13二元复合物，其表达质粒可以从Addgene库商购获得。将获得每种蛋白质的实验HDX-MS差异图以及这些输出与用于指导优化程序的天然组装体的模拟曲线之间的RMSE。在开发和优化该方法之后，它将被应用于了解葡萄球菌阻遏蛋白（Stl）和11 dUTR之间的组织。已经用一系列生物物理方法广泛表征了dUTPase-Stl相互作用，但是dUTPase-Stl组装的结构尚未成功结晶。通过分子对接已经产生了一系列潜在的复合物，但没有办法严格评估不同的对接姿势。在合作努力中，我们将获得dUTPase-Stl复合物的实验HDX-MS数据，然后应用我们的程序模拟候选组件的HDX-MS差异输出，并根据其与实验输出的一致性对其进行评估。