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Simulated isotope exchange patterns for protein structure determination

用于蛋白质结构测定的模拟同位素交换模式

基本信息

批准号：
BB/R006792/1
负责人：
Antoni Borysik
金额：
$ 28.21万
依托单位：
King's College London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2019
资助国家：
英国
起止时间：
2019 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FR006792%2F1
关键词：
Simulated isotope exchange patterns protein

项目摘要

Cell function is orchestrated by a large network of protein-protein interactions (PPIs) which in humans is thought to involve around 500,000 binary contacts between proteins. Almost every cellular process requires some form of PPI which could in theory be modulated a molecular therapeutic opening up new possibilities for drug development. Protein docking has emerged to meet the challenge of understanding the vast number of different PPIs at the molecular level. Docking is used to predict the structure of a protein assembly from the high resolution structures of its constituent monomers. While there have been many recent advances in protein docking extracting native assemblies from the high background of potential states that are typically generated remains a significant challenge. New approaches that allow improved ranking of protein docking outputs are required.In data-driven docking biophysical/biochemical aspects of protein structure are used as restraints to guide the selection of native conformations from protein docking simulations. Experimental outputs are typically simulated for each docking pose allowing them to be ranked according to the degree to which their simulated profiles agree to those obtained by experiment. There are many different types of experimental restraints that have been used to guide protein docking including those that report on protein size, shape and interaction strength. There are also a range of methods that provide direct insight into protein-protein interfaces and which offer enormous potential for data-driven docking. Nevertheless, leveraging the unique insights provided by these so-called footprinting methods has proven challenging.Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful footprinting and biophysical approach to study protein conformations. The technique reports on the change in mass accompanying the exchange of a protein's backbone amide protons for deuterium in D2O solvent resolved in peptide units of between 5 - 20 amino acids. For a binary PPI HDX-MS measurements are typically reported as difference plots between the bound and unbound proteins. These data provide direct insight into interacting protein regions as the isotope uptake at protein interfaces is slower in the assemblies due to solvent exclusion. HDX-MS is fast, sensitive, requires minimal sample preparation and is easy to automate and has the potential to revolutionise protein docking. However, the technique is currently limited to providing qualitative information on protein interfaces that cannot be used to critically evaluate different docking poses or to rank the simulated outputs.In this research I will develop methods to simulate HDX-MS data directly from protein docking outputs to allow high throughput and efficient ranking of whole docking simulations. I have provided significant primary results that clearly show the potential of this method to extract native structures from protein docking outputs. This approach is completely novel and also timely given the increased interest in HDX-MS due to its recent commercialisation. It will have applications across a broad base of bio-science and bio-pharmaceutical research and will represent a new way of working for practitioners of HDX-MS and molecular docking. Funding is required for development of the approach and optimisation of the codes so they are more suited for protein interfaces. The method will be written as a precompiled executable and be provided to researchers on request allowing them to simulate HDX-MS data from any protein docking outputs. The technique will also be applied to characterise the quaternary organisation between the Staphylococcal repressor protein (Stl) and the phi-11 dUTPase to shed new light on this important biological system.

细胞功能由蛋白质-蛋白质相互作用（PPI）的大型网络协调，在人类中，PPI被认为涉及蛋白质之间的约50万个二元接触。几乎每一个细胞过程都需要某种形式的PPI，理论上可以通过分子治疗来调节，为药物开发开辟新的可能性。蛋白质对接的出现是为了应对在分子水平上理解大量不同PPI的挑战。对接用于从蛋白质组成单体的高分辨率结构预测蛋白质组装体的结构。虽然最近在蛋白质对接方面取得了许多进展，但从通常产生的高背景势态中提取天然组装体仍然是一个重大挑战。在数据驱动的对接中，蛋白质结构的生物物理/生物化学方面被用作约束，以指导从蛋白质对接模拟中选择天然构象。实验输出通常模拟每个对接姿势，允许他们根据他们的模拟配置文件同意那些通过实验获得的程度进行排名。有许多不同类型的实验限制已被用于指导蛋白质对接，包括那些报告蛋白质大小，形状和相互作用强度。还有一系列方法可以直接洞察蛋白质-蛋白质界面，并为数据驱动的对接提供巨大的潜力。氢氘交换质谱（HDX-MS）是研究蛋白质构象的一种强大的生物物理学方法。该技术报告了伴随蛋白质的骨架酰胺质子交换为氘在D2 O溶剂中解析在5 - 20个氨基酸之间的肽单元中的质量变化。对于二进制PPI，HDX-MS测量通常报告为结合和未结合蛋白质之间的差异图。这些数据提供了对相互作用的蛋白质区域的直接洞察，因为在蛋白质界面处的同位素摄取在组装体中由于溶剂排斥而较慢。HDX-MS快速，灵敏，需要最少的样品制备，易于自动化，并有可能彻底改变蛋白质对接。然而，该技术目前仅限于提供定性信息的蛋白质接口，不能用于严格评估不同的对接姿势或排名的模拟outputs.In这项研究中，我将开发的方法来模拟HDX-MS数据直接从蛋白质对接输出，以允许高通量和有效的排名整个对接模拟。我已经提供了重要的初步结果，清楚地表明这种方法的潜力，从蛋白质对接输出提取天然结构。这种方法是完全新颖的，也是及时的，因为HDX-MS最近的商业化引起了人们对它的兴趣。它将在生物科学和生物制药研究的广泛基础上应用，并将代表HDX-MS和分子对接从业者的新工作方式。需要资金来开发方法和优化代码，使它们更适合蛋白质界面。该方法将被编写为预编译的可执行文件，并根据要求提供给研究人员，允许他们模拟来自任何蛋白质对接输出的HDX-MS数据。该技术也将被应用于研究葡萄球菌阻遏蛋白（Stl）和phi-11 dUTR之间的四级组织，以揭示这一重要的生物系统。