About the Project
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.
Please ask your referees to submit your references directly to Dr David Clarke [Email Address Removed].
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(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.
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