(BBSRC NWD) Harnessing fluorescent labels for novel in-cell biological structural determination using magnetic resonance

Understanding the structure of proteins is key to understanding their functions. Many structural biology methods involve sample preparation that both lengthy and takes proteins far away from their native conditions. In this project we will develop a novel photo-activated family of radical-based spin-labels for use in Electron Paramagnetic Resonance Pulsed Dipolar Spectroscopy (EPR-PDS) and Nuclear Magnetic Resonance Paramagnetic Relaxation Enhancement (NMR-PRE) experiments.[1] These methods determine distances on a nanometre scale within protein structures. The proposed radical-based spin-labels are formed on rhodamine and xanthene dyes, commonly used in Förster Resonance Energy Transfer (FRET), using photo-initiated chemistry under reducing conditions.[2] There are several potential advantages of these labels over existing radical labels, firstly they are activated by light and therefore measurements recorded in the dark can be used as reference data for example in NMR-PRE where the relaxation enhancement is measured relative to the sample in the absence of the radical. Also, the formation of the radical takes place in reducing conditions and this reveals the possibility of using such labels for in-cell EPR-PDS measurements, conditions where typical radical labels are not stable.  

Initially we will benchmark the novel labels against the more commonly used nitroxide based labels using calmodulin as a test system, already extensively studied using EPR-PDS and NMR-PRE with nitroxide labels.[3] FRET experiments will be performed to further characterise the system.[4] We will make use of a novel freeze-quench that allows time resolution of processes to be analysed, and we will integrate a light source into this apparatus allowing samples to be frozen under illumination. Once the methodologies have been optimised on the calmodulin, we will apply the novel techniques to less well understood protein systems including mechano-sensitive ion channels.[5] 

Dr Bowen has extensive experience in magnetic resonance methods, including optically activated EPR and NMR methods, and will both oversee the project and supervise these measurements.  We will make use of the NMR torch methodology, developed by Dr Golovanov, to allow in-situ illumination of the sample so both experiments can be performed without removing the sample. Prof. Hay has extensive experience working with Calmodulin and integrating distance based restrains into models of this system. Dr Heyes will supervise optical characterization the excited states of the dyes used and FRET to extract inter-dye distances. Dr Pilotas will oversee the preparation of in-cell samples and the work on mechano-sensitive ion channels, in which he is an expert. 

For informal enquiries prior to application please contact Dr Alice Bowen: alice.bowen@manchester.ac.uk 

Dr Alice Bowen: https://research.manchester.ac.uk/en/persons/alice.bowen 

Prof. Sam Hay: https://research.manchester.ac.uk/en/persons/samhay-manchesteracuk-hay 

Dr Derren Heyes: https://research.manchester.ac.uk/en/persons/derren.heyes 

Dr Alexander Golovanov: https://research.manchester.ac.uk/en/persons/a.golovanov 

Dr Christos Pliotas: https://research.manchester.ac.uk/en/persons/christos-pliotas 

The National Research Facility for Electron Paramagnetic Resonance:  https://www.chemistry.manchester.ac.uk/epr/ 

Eligibility

Applicants must have obtained or be about to obtain a minimum Upper Second class UK honours degree, or the equivalent qualifications gained outside the UK, in an appropriate area of science, engineering or technology. 

International applicants

We are only able to offer a limited number of full studentships to applicants outside the UK. Therefore, full studentships will only be awarded to exceptional quality international candidates due to the competitive nature of this scheme.

International applicants must ensure they meet the academic eligibility criteria (including English language) before applying. Visit our ‘Eligibility and entry requirements’ page to find out more about our English language requirements.

Before you Apply

Browse our BBSRC NWD in Bioscience projects and discover one you’re passionate about that matches your interests, ambitions and goals.

Applicants must make direct contact with preferred supervisors before applying. It is your responsibility to make arrangements to meet with potential supervisors, prior to submitting a formal online application.

How to Apply

All applications should be made via our specific BBSRC NWDP in Bioscience online application form. This is a separate application process specifically for all candidates for this scheme. Applications should not be made through either university’s usual online application system.

You must submit your application form along with the required supporting documents by the deadline date. You can select up to two projects on one single application, noting the title of each project from the advert and the supervisor name. This can include two projects from one institution or a project from each institution. 

Before you complete your application you should prepare the following supporting documents:

·            CV

·            Personal statement

·            Degree certificates and transcripts (if your qualification is still pending, please ensure this is clear on your application form and you can send this to us at a later date)

·            Two references

·            Evidence of English language qualifications (if completed)

Complete the application form: https://forms.office.com/e/HVXALrVa0P

Once you have completed your application, you’ll receive a confirmation email and             you should reply to this email with all of your supporting documentation as soon as possible to: nwd-bbsrc.applications@manchester.ac.uk

Deadline: Friday, 31 January 2025, 5pm

Late or incomplete applications will not be considered.

If you need help with this stage of the process, or have any queries regarding your eligibility (such as if you achieved unexpectedly low degree results due to extenuating circumstances), please contact the Doctoral Admissions team for advice at fbmh.doctoralacademy.admissions@manchester.ac.uk. 

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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.