Simulated isotope exchange patterns for protein structure determination

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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.