Designer cross-linking chemistry to probe protein-protein interactions in cells

Supervisor(s)

Contact Professor Andrew J Wilson to discuss this project further informally.

Project description

The goal of this project is to develop covalent cross-linking reagents that possess (1) suitably reactive groups for high-yielding cross-linking over a variety of timescales and (2) handles (fluorophores, affinity groups) for analyses in cells. We will then use these reagents to study the interactome of outer membrane proteins (OMPs) the beta-barrel assembly machinery (BAM) and relevant chaperones of Gram negative bacteria. A key problem in life-sciences research is to understand cellular processes with molecular and temporal resolution – this would allow the identification of the transient intermediates that play key roles in the function of biomacromolecular machines, signalling, translocation and folding.

We recently developed tag-and-transfer diazirine-based reagents and, a rapid photochemical reactor that can be used to interrogate protein binding and conformational changes on a rapid timescale. In this research we will further engineer these tag-and-transfer diazirine reagents so they can be incorporated into proteins using genetic methods (i.e. Amber Suppression). We will then use the developed reagents to elucidate the mechanism of biogenesis of the outer membrane of E.coli, focusing on BAM-dependent catalysis of the folding and membrane insertion of outer membrane proteins (OMPs). The results will open the door to new methods for delineating molecular reactions in cells, in general, as well as to elucidate how OMPs fold- a question of critical importance and utility in the drive to develop new antimicrobial agents that target this pathway.

The student will gain skills in: (i) synthetic chemistry through design and synthesise of tag-and transfer amino-acid diazirines bearing additional orthogonal reactive groups (e.g. click reagents to permit addition of biotin for pull-down) (ii) molecular biology approaches to identify appropriate tRNA/Synthetase pairs that accept the novel amino acids and incorporate these into OMPs, components of BAM, and relevant chaperones; and (iii) cross-linking, isolation and characterization of their protein complexes, which will be analysed using mass spectrometry.

This project is eligible for funding under the BBSRC Doctoral Training Programme to start in October 2019 – applications close on 7th Jan 2019. Please contact Prof. Andy Wilson (A.J.Wilson@leeds.ac.uk) for further details about this opportunity.

References 1. J. M. Fletcher, K. A. Horner, G. J. Bartlett, G. G. Rhys, A. J. Wilson, D. N. Woolfson: De novo coiled-coil peptides as scaffolds for disrupting protein-protein interactions Chem. Sci., 2018, 9, 7656-7665. 2. C. M. Grison, G. M. Burslem, J. A. Miles, L. K. A. Pilsl, D. J. Yeo, Z. Imani, S. L. Warriner, M. E. Webb, A. J. Wilson: Double Quick, Double Click Reversible Peptide “Stapling”, Chem. Sci., 2017, 8, 5166-5171. 3. G. Preston, S. E. Radford, A. J. Wilson: Analysis of Amyloid Nanostructures Using Photo-Crosslinking: In Situ Comparison of Three Widely Used Photo-Crosslinkers, ACS Chem. Biol., 2014, 9, 761–768 4. M.G. Iadanza, A. J. Higgins, R. Schiffrin, A. N. Calabrese, D.J. Brockwell, A.E. Ashcroft, S. E. Radford, N. A. Ranson: Lateral opening of the intact 1 β-barrel assembly machinery captured by cryo-EM, Nature Comms., 2016, 7, 12865. 5. R. Schiffrin, A.N. Calabrese, P. W. A. Devine, S. A. Harris, A. E. Ashcroft, D. J. Brockwell, S. E. Radford: Skp is a multivalent chaperone of outer membrane proteins, Nat. Struct. Mol. Biol. 2016, 23, 786-793.

For further information download the PhD opportunity advert.

Entry requirements

Candidates should have, or be expecting, at least a 2:1 honours degree or equivalent. We welcome students with backgrounds in biological, chemical or physical sciences, or mathematical backgrounds with an interest in biological questions. 

If English is not your first language, you must provide evidence that you meet the University’s minimum English Language requirements.

Additional staff contact

Prof Sheena E Radford FRS

How to apply

Formal applications for research degree study should be made online through the university's website. Please state clearly in the research information section that the PhD you wish to be considered for is the 'Designer cross-linking chemistry to probe protein-protein interactions in cells’ as well as Professor Andy Wilson as your proposed supervisor.

We welcome scholarship applications from all suitably-qualified candidates, but UK black and minority ethnic (BME) researchers are currently under-represented in our Postgraduate Research community, and we would therefore particularly encourage applications from UK BME candidates. All scholarships will be awarded on the basis of merit.

If you require any further information please contact the Graduate School Office e: maps.pgr.admissions@leeds.ac.uk