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In situ HDX-MS of membrane protein-ligand interactions


   Faculty of Biology, Medicine and Health

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  Dr A Politis  No more applications being accepted  Funded PhD Project (Students Worldwide)

About the Project

The project aims to develop an HDX-MS workflow to profile ligand-membrane protein interactions. The identification of hits with high selectivity constitutes an important landmark for the design and development of novel therapeutics. Traditional strategies such as cell-based assays often identify upstream targets modifying protein function indirectly, whereas structure-based drug design is limited by the lack of protein structures from human species. These limitations underscore the need for new strategies, which can map membrane protein-drug interactions even in the absence of structure. To date, the application of HDX-MS for ligand screening remains low-throughput, owing to the lack of methods and instrumental capabilities. For instance, limitations in peak capacity and chromatography often result in inefficient separation and low quality data. To overcome such limitations and to build a novel platform for biophysical characterisation of membrane proteins in situ we will combine HDX-MS of membrane proteins with IM-MS separations.

Excitingly, in this project will carry out in-situ HDX-IM-MS in cell membranes. We will target transporters and receptors, overexpressed from human cells and solubilised in lipid vesicles. Proteins will be incubated with drugs/ligands and analysed by HDX-IM-MS, reporting on binding site(s) and allosteric changes. We will compare the results with those obtained in detergents, nanodiscs and/or liposomes, establishing a comprehensive understanding on the impact of native cellular environment on protein dynamics. To gain in speed of analysis, we will optimise conditions in digestion and LC for covering the binding pocket and allosteric sites. We will scale up the approach to screen multiple ligands, using multiplexes of compounds. Finally, we could obtain residue-level resolution by selecting key peptides for fragmentation by ETD capabilities. Overall, this project will build a new strategy and methodology to identify membrane protein-ligand interactions in the native environment where they reside.

Entry Requirements

Applicants must have obtained or be about to obtain a First or Upper Second class UK honours degree, or the equivalent qualifications gained outside the UK in Biochemistry, Biological Sciences, Chemistry or a related discipline. A Master's degree in a relevant subject and / or experience in the above related areas is desirable.

Applicants interested in this project should make direct contact with the Primary Supervisor to arrange to discuss the project further as soon as possible.

How To Apply

To be considered for this Studentship you MUST submit a formal online application form - full details on how to apply can be found on https://www.bmh.manchester.ac.uk/study/research/apply/

Equality, Diversity and Inclusion

Equality, diversity and inclusion is fundamental to the success of The University of Manchester, and is at the heart of all of our activities. The full Equality, diversity and inclusion statement can be found on the website https://www.bmh.manchester.ac.uk/study/research/apply/equality-diversity-inclusion/


Funding Notes

Studentship funding is for a duration of 3.5 years to commence in September 2022 and covers UKRI equivalent fees and stipend (£16,062 per annum 22/23)
Funding will cover UK tuition fees and stipend only. The University of Manchester aims to support the most outstanding applicants from outside the UK. We are able to offer a scholarship that will enable a full studentship to be awarded to international applicants. This full studentship will only be awarded to exceptional quality candidates, due to the competitive nature of this funding.

References

1. Martens, C.; Shekhar, M.; Lau, A.M.; Tajkhorshid, E., Politis, A*. Integrating hydrogen-deuterium exchange mass spectrometry with molecular dynamics simulations to probe lipid-modulated conformational changes in membrane proteins. Nature Protocols, 14, 3183–3204, 2019
2. Jia R, Martens C, Shekhar M, Pant S, Pellowe GA, Lau AM, Findlay HE, Harris NJ, Tajkhorshid, Booth PJ, Politis A*. Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter. Nature Communications, 11: 6162, 2020
3. Ahdash, Z.; Pyle E.; Allen, W.J.; Corey, R.A.; Collinson, I. Politis, A*. HDX-MS reveals nucleotide-based, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon. eLife, e47402, 2019
4. Martens C, Shekhar M, Borysik A, Reading E, La AM, Tajkorshid E, Booth PJ, Politis A*. Direct protein-lipid interactions shape the conformational landscape of secondary transporters. Nature Communications, 9:4151 2018
5. Yen H-Y, Liko I, Song W, Kapoor P, Almeida F, Toporowska J, Gherbi K, Charlton S, Politis A, Sansom M, Jazayeri A, Robinson C. Mass spectrometry captures biased signalling and allosteric modulations of a G-protein coupled receptor. Under review, Nature Chemistry
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