Dissecting the Campylobacter jejuni flagellar motor using subtomogram averaging and rationally designed protein chimeras
File(s)
Author(s)
Henderson, Louie Derek
Type
Thesis or dissertation
Abstract
Bacterial cells must traverse a variety of environments to survive and thrive. To
achieve motility, they have evolved the bacterial flagella motor. This complex proteinacious nano-machine is composed of up to 25 different proteins, which assemble
together in a range of stoichiometries across multiple membranes. They are able to
harness the flux of ions across inner membrane bound stators, to kick a cytoplasmic
C-ring, thus generating torque. Rotational torque is then transmitted through the
periplasmic axle, which can be augmented by additional protein scaffolds, to the
external hook and filament, propelling the cell forwards. Through developments of
in situ Electron Cryo-Tomography (ECT), increased structural complexity in the
motors of species such as Campylobacter jejuni have been explored to nanometer
resolutions. This has revealed additional, intricate complexes which enable increased
base and novel functions.
Using the C. Jejuni motor as a model, I have applied and developed a number of
methods to study its complexity, in hopes of understanding the structures of functional proteinacious nanomachines in their native context. By dissecting the in situ
structure of the cytoplasmic C. jejuni C-ring, I have revealed the domain placements of FliG C terminal, FliM, FliN and FliY. Combining this with phylogenetic
and operon analysis, I have also explored the evolved structural role of FliNY intercalation at the distal tip of the C-ring, revealing the greater role FliY plays in
the structural anchoring of the ATPase complex to the C-ring in C. jejuni. Furthermore, I’ve characterised a previously unknown component of the periplasmic scaffold
dubbed Mot1, which acts as a stabilising outer brace by anchoring the proximal and
sub-basal disks. To overcome the resolution limitations of ECT, which prevent unambiguous identification of protein components in nano-machines, I’ve developed
software which identifies protein chains to insert into macromolecules without disrupting tertiary or quaternary structure to act as electron dense tags.
achieve motility, they have evolved the bacterial flagella motor. This complex proteinacious nano-machine is composed of up to 25 different proteins, which assemble
together in a range of stoichiometries across multiple membranes. They are able to
harness the flux of ions across inner membrane bound stators, to kick a cytoplasmic
C-ring, thus generating torque. Rotational torque is then transmitted through the
periplasmic axle, which can be augmented by additional protein scaffolds, to the
external hook and filament, propelling the cell forwards. Through developments of
in situ Electron Cryo-Tomography (ECT), increased structural complexity in the
motors of species such as Campylobacter jejuni have been explored to nanometer
resolutions. This has revealed additional, intricate complexes which enable increased
base and novel functions.
Using the C. Jejuni motor as a model, I have applied and developed a number of
methods to study its complexity, in hopes of understanding the structures of functional proteinacious nanomachines in their native context. By dissecting the in situ
structure of the cytoplasmic C. jejuni C-ring, I have revealed the domain placements of FliG C terminal, FliM, FliN and FliY. Combining this with phylogenetic
and operon analysis, I have also explored the evolved structural role of FliNY intercalation at the distal tip of the C-ring, revealing the greater role FliY plays in
the structural anchoring of the ATPase complex to the C-ring in C. jejuni. Furthermore, I’ve characterised a previously unknown component of the periplasmic scaffold
dubbed Mot1, which acts as a stabilising outer brace by anchoring the proximal and
sub-basal disks. To overcome the resolution limitations of ECT, which prevent unambiguous identification of protein components in nano-machines, I’ve developed
software which identifies protein chains to insert into macromolecules without disrupting tertiary or quaternary structure to act as electron dense tags.
Version
Open Access
Date Issued
2019-12
Date Awarded
2020-06
Copyright Statement
Creative Commons Attribution NonCommercial Licence
Advisor
Beeby, Morgan
Buck, Martin
Sponsor
Biotechnology and Biological Sciences Research Council (Great Britain)
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)