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EASTBIO Understanding Protein Nanocompartment Assembly for Efficient Engineering of Encapsulated Enzymes


School of Chemistry

Dr D Clarke , Wednesday, January 06, 2021 Competition Funded PhD Project (Students Worldwide)

About the Project

The supervisory team of this project merges expertise in enzymology (DC/SW), biological mass spectrometry (DC) and synthetic biology (SW). This project will provide training in state-of-the-art scientific research and analytical methods and will ensure the acquisition of skills in the areas of protein chemistry, enzymology, structural biology and biological mass spectrometry.

Encapsulin nanocompartments are spherical bacterial capsid-like compartments roughly 25-30 nm in diameter. These large protein assemblies are made up of either 60 or 180 copies of a single 30 kDa protomers which assemble to form a hollow icosahedral protein cage. A partner enzyme is sequestered inside the encapsulin by an interaction between a short encapsulation sequence (ES) appended to the protein and the interior of the encapsulin shell. The resulting complex offers a privileged environment for performing catalysis and a store for potentially harmful products or intermediates in enzymatic pathways (1).

A number of different types of enzymes have been identified as encapsulin cargo-proteins – including peroxidases and ferritins. However, recent work has demonstrated that the ES sequence can be grafted onto heterologous proteins to localise them to the lumen of the encapsulin, and a number of heterologous reporter proteins have been directed to encapsulins in this way. This ability to encapsulate non-native proteins, coupled with the potential to engineer the outer-surface and pores of the encapsulin shell, affords these systems great potential as nano-machines for biocatalysis, bioremediation, and as therapeutic tools. However, little is currently known about the loading stoichiometry of native or heterologous cargo proteins within encapsulins; how these complex systems assemble in vivo; or how loading stoichiometry effects the enzyme activity and the storage capacity of these systems. Understanding the structure, biochemistry and biophysics of these systems is required in order to fully realise their potential as powerful nanotechnology and synthetic biology platforms.

In this PhD project the student will produce a series of native and heterologous encapsulated systems using an established modular synthetic biology platform. In collaboration with Steven Wallace, a series of industrial biocatalytic target enzymes will be chosen for encapsulation and the effect of encapsulation on stability and activity will be determined. The assembly of these encapsulated systems will then be monitored using a combination of several structural mass spectrometry approaches. Native mass spectrometry and ion mobility mass spectrometry techniques allow the observation and characterisation of intact encapsulin systems (2). By combining this technology with rapid mixing techniques, developed in the Clarke lab (3), we will observe the stepwise assemble of encapsulin systems. These studies will outline the parameters for efficient encapsulation of proteins in these systems– providing a framework for the production of designed encapsulated enzymes.

The student will be trained in synthetic biology techniques, protein production, purification and biochemistry. In addition, the student will gain hands-on experience in structural mass spectrometry and will develop new structural mass spectrometry techniques. Experiments will be performed on the newly installed BBSRC-funded state-of-the-art high field Fourier transforms ion cyclotron resonance mass spectrometer (FT-ICR MS) housed at the School of Chemistry.

Application Process:
To apply for an EASTBIO PhD studentship http://www.eastscotbiodtp.ac.uk/, follow the instructions below:
Check FindaPhD https://www.findaphd.com/phds/program/bbsrc-eastbio-doctoral-training-partnership-call-for-applications-for-2021/?p1048 for our available projects and contact potential supervisors before you apply.

After you have discussed the projects of interest to you with the project supervisors, download and complete our Equality, Diversity and Inclusion survey https://edinburgh.onlinesurveys.ac.uk/eastbio-dtp-equality-diversity-inclusion-form-2021 and then fill in the EASTBIO Application Form and submit the application form plus your academic transcripts to Dr David Clarke () (Links to the forms can be found here: http://www.eastscotbiodtp.ac.uk/how-apply-0)

Send the EASTBIO Reference Form to your two academic/professional referees, and ask them to submit these directly to Dr David Clarke () (Link to the form can be found here: http://www.eastscotbiodtp.ac.uk/how-apply-0)

If you are nominated by the supervisor(s) of the EASTBIO PhD project you wish to apply for, they will provide a Supervisor Support Statement.

All EASTBIO (online) interviews will be in the week 8-12 February 2021 with awards made the following week.

The School of Chemistry holds a Silver Athena SWAN award in recognition of our commitment to advance gender equality in higher education. The University is a member of the Race Equality Charter and is a Stonewall Scotland Diversity Champion, actively promoting LGBT equality. The University has a range of initiatives to support a family friendly working environment. See our University Initiatives website for further information. University Initiatives website: https://www.ed.ac.uk/equality-diversity/help-advice/family-friendly

Funding Notes

This 4 year PhD project is part of a competition funded by EASTBIO BBSRC Doctoral Training Partnership View Website. This opportunity is open to UK and International students and provides funding to cover stipend and tuition fees. Please refer to UKRI website View Website and Annex B View Website of the UKRI Training Grant Terms and Conditions for full eligibility criteria. Applicants must have a good first degree (minimum of 2.1) in an appropriate Biological Science.

References

References
(1) R. J. Nichols, et al. Critical Reviews in Biochemistry and Molecular Biology. 2017 52 (5): 583–594.
(2) J. Snijder et al. J. Am. Chem. Soc.2014 136, 20, 7295-7299
(3) D. J. Clarke, et al. Anal. Chem. 2010, 82, 1897-1904.


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