Summary:
During this PhD project you will use ion mobility mass spectrometry to investigate the conformations of ubiquitin (Ub) shuttle proteins. The conformations of these proteins that form dense protein-rich droplets within cells will be investigated, as well as their interactions with additional binding partners. You will become an expert in ion mobility mass spectrometry, analytical chemistry, protein biophysics, protein degradation pathways and biomolecular interactions. You will present your research at conferences and undergo a broad training program on research-related and transferable skills. The project will be based in the lab of Dr Rebecca Beveridge, which houses excellent IMMS infrastructure.
Background:
Intrinsically disordered proteins (IDPs) exist and function without the fixed tertiary structure that was once thought to be required for all proteins to carry out their physiological roles, and instead populate a wide range of conformations, from compact to extended. IDPs and intrinsically disordered regions (IDRs) in proteins are an important focus of research due to their high abundance and overrepresentation in disease states. Additionally, long IDRs often contribute to liquid-liquid phase separation (LLPS) which results in the formation of dense droplets containing protein and other biomolecules. LLPS is important in normal cell physiology, and dysfunctional LLPS is involved in diseases such as Alzheimer’s Disease and amyotrophic lateral sclerosis. Investigations into the conformational changes of IDPs as they undergo LLPS has remained challenging, as they remain elusive to most biophysical techniques.
We have recently used IMMS to investigate the conformations of the protein Ubiquilin-2 (UBQLN2) as it undergoes LLPS. LLPS of UBQLN2 occurs in response to increases in salt concentration, and is reversed and inhibited by the binding of ubiquitin (Ub). We therefore measured the conformational distributions of UBQLN2 in its soluble form at low salt concentration, delineated its conformational response to increased salt concentrations that drive LLPS, and interrogated the complexes it forms with Ub that reverse and inhibit LLPS. We identified that UBQLN2 exists as a mixture of monomers and dimers, both with a wide range of conformations. At increased salt concentration, the dimers undergo a subtle shift to more extended conformations that we hypothesise are implicit in driving LLPS. In contrast, the presence of Ub stabilises compact conformations of UBQLN2 dimers which we propose are unable to form the multivalent intermolecular interactions required for LLPS.
In the proposed research we will apply these newly developed methods that have shed important light on UBQLN2 to a suite of additional proteins to identify fundamental rules of ubiquitin controlled LLPS. The research will aim to answer the following questions:
(1) How do LLPS mechanisms compare across Ub shuttle proteins?
(2) What are the differences in LLPS mechanisms in healthy and disease states?
(3) How can ion mobility mass spectrometry be better developed to investigate these proteins?
If you wish to discuss any of the details informally, please contact Dr Rebecca Beveridge ([Email Address Removed]).
Entry Requirements:
A very good undergraduate degree (at least a UK 2:1 honours degree, or its international equivalent).
How to apply:
Please send your C.V., a cover letter and details of two referees to Dr Beveridge ([Email Address Removed]). The cover letter should outline why you’re interested in this project and describe your relevant experience.