Development of global analysis tools to model protein isotope exchange behaviour

Lead Research Organisation: King's College London
Department Name: Chemistry

Abstract

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.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M014851/1 01/10/2015 28/02/2021
1862995 Studentship BB/M014851/1 01/11/2016 31/05/2020 Matthew Harris