Mass photometry enabled label-free platform to study the dynamics of membrane proteins assembly

This studentship is supported by the Aston Institute for Membrane Excellence.

Integral membrane proteins (IMPs) are crucial for cell functions including nutrient uptake, cell-fate regulation, signalling, water and small molecule transport, neuronal transduction and migration. In addition, IMPs are critical to regulate the tissue and organ patterning and in maintenance of physiological states in adults. MPs role in cell physiology maintenance is outlined by their significant role in viral infection, various physiological diseases such as Diabetes, Alzheimers, Parkinsons, Cystic Fiborosis, and in a range of cancers. Thus, it is not surprising to see that significant number of drugs target IMPs.

IMPs function is intrinsically linked to their oligomeric nature and interactions with other IMPs and soluble proteins, hence, understanding IMP function and its dysregulation requires in-depth understanding of IMP oligomeric nature as well as their interaction pattern in case of health and diseased condition. Studying IMPs, however, remains extremely challenging due the unique presence of hydrophobic trans-membrane domain (TMD) that requires hydrophobic environment to remain stable in otherwise aqueous environment in the cell. In cells, IMPs are inserted through the membrane bilayer to stabilise and maintain their function. Moreover, lipid composition and the height of membrane bilayers is crucial to maintain IMP oligomeric nature and function.

A large number of alternates to membrane bilayers, including detergents, SMALPs, nanodiscs, peptidiscs have been devised to solubilise and stabilise IMPs, however, significant efforts are needed to maintain the functional state, including oligomeric nature, of IMPs. Moreover, estimating oligomeric nature of the IMP still remains extremely difficult and majorly carried out via time consuming native mass spectrometry, X-ray crystallography, and Cryo-EM.

Mass photometry (MP), a label-free imaging technique is well suited to study the oligomeric nature of proteins both in solution and on membrane, termed as dynamic MP. Preliminary results with the reconstitution membrane pore protein in bilayers suggest that dynamic MP can be used to track both the oligomeric nature as well as the diffusion coefficient of a pore protein inserted into the bilayer. This opens up the possibility to study dynamics of IMP proteins assembly and the effect of bilayer properties including lipids composition, fluidity on IMP dynamics.

This project will employ an interdisciplinary approach where several techniques and approaches, including microfluidics, polymer chemistry, glass surface modification, membrane deposition, membrane protein expression-purification, image-analysis and single-particle-tracking will be employed.

This work will involve development of a mass photometry and lipid bilayer compatible polymeric layer deposition on glass and methodologies to deposit lipid-bilayers on the polymeric cushion (cushioned bilayers).

We will use fluorescence microscopy and single-particle-tracking to compare the lipid fluidity in cushioned bilayers and glass supported bilayers. These standardised cushioned bilayers will be used to develop generalised strategies to insert and assemble IMPs in the cushioned bilayers. For this purpose, we will use pore proteins (e.g. cytolysins, haemolysin, and Shiga Toxins) and membrane proteins (e.g. aquaporins) with known oligomeric nature to standardize the insertion methodologies. The oligomeric assembly and diffusion of pore/IMPs will be estimated using Mass photometry enabled single-particle-tracking. The methodology will be validated by changing lipid composition and by addition of protein/ small molecule that can disrupt pore/IMP assembly and thus providing a method to not only study the IMP oligomeric nature, but also understand the conditions that can disrupt IMP assembly opening avenues to design/test drugs against clinically significant IMPs.

During the PhD, the candidate will be supported by the interdisciplinary team at AIME and will be working with the leading edge of technologies including membrane protein research and single-particle-tracking using mass photometry at AIME. The project will provide excellent skills to the student to proceed their career both in academics and in industry.

The successful applicant should have been awarded, or expect to achieve, a Masters degree in a relevant subject with a 60% or higher weighted average, and/or a First or Upper Second Class Honours degree (or an equivalent qualification from an overseas institution) in a relevant subject. Good theoretical knowledge of biochemistry, microscopy, enzyme kinetics, lipid bilayers. Practical experience with microbiology and protein expression and purification is desirable but not necessary. Knowledge of coding would be beneficial. 

For enquiries contact Manish Singh Kushwah at m.kushwah@aston.ac.uk

We can only consider applications that are complete and have all supporting documents. Applications that do not provide all the relevant documents will be automatically rejected. Your application must include:

1. English language copies of the transcripts and certificates for all your higher education degrees, including any Bachelor degrees.
2. A Research Statement detailing your understanding of the research area, how you would approach the project, and a brief review of relevant literature. Be sure to use the title of the research project you are applying for. There is no set format or word count.
3. A personal statement which outlines any further information which you think is relevant to your application, such as your personal suitability for research, career aspirations, possible future research interests, and further description of relevant employment experience.
4. Two academic referees who can discuss your suitability for independent research. References must be on headed paper, signed and dated no more than 2 years old. At least one reference should be from your most recent University. You can submit your references at a later date if necessary.
5. Evidence that you meet the English Language requirements. If you do not currently meet the language requirements, you can submit this at a later stage.
6. A copy of your passport. Where relevant, include evidence of settled or pre-settled status.

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Please select “ Research – Biomedical Sciences” from the application form options.