Biophysical characterisation of the Campylobacter jejuni β-barrel assembly machinery (BAM) complex mediated outer membrane transport pathway
Publication Date
July 18, 2023
Creator
Abstract
Outer membrane proteins in Gram-negative bacteria perform many functions that are key to bacterial survival, extracellular transport, and virulence. In order for these proteins to get to the outer membrane where they perform their function, they engage in a transport pathway starting from the ribosome, traversing the inner membrane to the periplasm where they are folded and assembled into the outer membrane. Several transport pathways contribute to this final folding and assembly step, however, it was recently established that the majority of outer membrane folding and assembly in Gram-negative bacteria is attributed to the β-barrel assembly machinery (BAM) complex. This PhD aims to extend the present knowledge on the BAM complex through biophysical and computational characterisation of the key proteins involved in the BAM-mediated transport pathway of Campylobacter jejuni.
This thesis describes the preparation of C. jejuni BamA, BamD, Cj1289, DegP and LptD from cloning through to expression and purification, and subsequent experimental characterisation using circular dichroism and small angle X-ray scattering where folding, melting temperatures, complexation and protein-protein interactions are analysed. In tandem with the experimental wet work, homology models were generated and in silico docking runs were used to analyse the docking interface between BamA and BamD, and to prepare models for molecular dynamics (MD) simulations. Further experiments in the pipeline for elucidating structural insights through cryogenic transmission electron microscopy and MD simulations are briefly described.
The results presented in this work suggest three main findings. Firstly, Landau free energy expansion models may provide more accurate melting temperature estimations than the current Boltzmann analysis methodology. Secondly, that there is an interaction between Cj1289 (SurA-like chaperone) and BamD not previously cited in the literature. Thirdly, given knowledge of an approximate binding position, MD simulations may be a potential alternative to rigid docking that provides additional information on docking site and conformational sampling occurring prior to the final expected binding position.
Item Type
ethesis
Thesis Type
PhD
Supervisors
Subjects (LC)
Associated Schools / Departments
School of Life Sciences
eprints ID
73682
UoN Repository URI
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