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EastBio Understanding the Structural Basis of Encapsulin Assembly for Efficient Engineering of Encapsulated Enzymes

  • Full or part time
    Dr D Clarke
  • Application Deadline
    Sunday, January 05, 2020
  • Competition Funded PhD Project (European/UK Students Only)
    Competition Funded PhD Project (European/UK Students Only)

Project Description

Co-Supervisors: Dr Stephen Wallace, School of Biological Sciences, University of Edinburgh. Dr. Louise Horsfall, School of Biological Sciences, University of Edinburgh

Project Details: 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 protomer 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 (2). 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. Understanding the structure, biochemistry and biophysics of encpasulins 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 by recombinant expression in E. coli. 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 (3). By combining this technology with rapid mixing techniques (4), 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:
http://www.eastscotbiodtp.ac.uk/how-apply-0
Please ask your referees to submit your references directly to Dr David Clarke .

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:

Funding Notes

This 4 year PhD project is part of a competition funded by EASTBIO BBSRC Doctoral Training Partnership View Website. This opportunity is only open to UK nationals (or EU students who have been resident in the UK for 3+ years immediately prior to the programme start date) due to restrictions imposed by the funding body.

References

(1) R. J. Nichols, et al. Critical Reviews in Biochemistry and Molecular Biology. 2017 52, 583–594.
(2) D. He, S. et al. eLife. 2016. 5, e18972.
(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.

How good is research at University of Edinburgh in Chemistry?
(joint submission with University of St Andrews)

FTE Category A staff submitted: 43.30

Research output data provided by the Research Excellence Framework (REF)

Click here to see the results for all UK universities

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