Multimodal cryoEM approaches enable reaching sub-nanometre resolutions in situ and modelling of known components in a high-torque flagellar motor
Lead Research Organisation:
Imperial College London
Department Name: Life Sciences
Abstract
Understanding how evolutionary mechanisms gave rise to contemporary cellular pathways and protein complexes is key in understanding the development of life on earth. However, as there is no fossil record to provide glimpses of their ancestral forms, we are limited to deducing ancestral intermediates from observing contemporary variation.
The bacterial flagellar motor is a great case study for understanding evolution of macromolecular complexes. It enables swimming by rotating a flagellar filament and its mechanistic core is conserved across all bacteria. However, different bacterial clades evolved divergent higher-torque motors by incorporating additional protein components on top of the conserved core. This high degree of variation makes the flagellar motor well-suited for studying how new proteins are incorporated into existing macromolecular machines.
Campylobacter jejuni and related epsilon-proteobacteria have complex high-torque motors and will serve as models for me to study evolution on a molecular scale. I am initially focussing on two epsilon-proteobacterial proteins, PflA and PflB, building on previous work from our lab. PflAB form periplasmic disk structures, thought to function as scaffolding by interacting with other motor components, recruited by an ancestor of epsilon-proteobacteria.
Where did additional proteins, such as PflAB, come from and how did they initially associate with the motor? What changes enabled them to be integral to the motor? What is the order of incorporation of additional components? My approach to tackle these questions is threefold.
Firstly, in situ cryo electron-tomography and subtomogram averaging will yield low-to-intermediate resolution contextual information. Imaging bacterial mutants with a deleted protein of interest helps determine its location within the motor. Earlier work by collaborators identified two proteins, essential for C. jejuni motility. To help understand their functions and determine if they are part of the periplasmic scaffold, I will image their deletion mutants. Imaging other relevant bacteria will help build the motors evolutionary history. Bdellovibrio bacteriovorus is a related bacterium with a potential distant PflA homolog. Determinig the motor structure of this deletion mutant will help establish if the protein is a PflA homolog, and whether the motor is descended from an early precursor before incorporation of other accessory proteins.
Secondly, in vitro work will provide higher resolution information on protein domains and atomic structure. To become part of a complex, a protein must evolve a binding interface. To define protein binding interfaces between periplasmic flagellar proteins (e.g. PflAB, MotB), I will perform pull-down assays for protein pairs, believed to interact, using different truncations to identify interacting regions. Additionally, I will attempt to obtain atomic structures of purified proteins and protein complexes by combination of X-ray crystallography and single particle analysis.
Finally, in silico phylogenetics and sequence analysis will complement wet lab research. To build an evolutionary history of epsilon-proteobacterial motors and help determine order of recruitment of peripheral proteins, we will build a phylogeny of accessory proteins and compare with phylogeny of core flagellar components. PflAB contain many tandem TPR motifs, repeats often present in protein-binding interfaces. We will tpredict PflAB protein-binding regions, comparing sequences between motifs and from dn/ds ratios (rates of non-synonymous and synonymous mutations), which will enhance our understanding of the functional mechanisms of PflAB in the motor.
These sets of structural, bioinformatic, phylogenetic, and molecular biological experiments will be highly informative about the role of these proteins in the flagellar motor, how they perform these functions, their evolutionary mechanisms and path to incorporation, contributing to our
The bacterial flagellar motor is a great case study for understanding evolution of macromolecular complexes. It enables swimming by rotating a flagellar filament and its mechanistic core is conserved across all bacteria. However, different bacterial clades evolved divergent higher-torque motors by incorporating additional protein components on top of the conserved core. This high degree of variation makes the flagellar motor well-suited for studying how new proteins are incorporated into existing macromolecular machines.
Campylobacter jejuni and related epsilon-proteobacteria have complex high-torque motors and will serve as models for me to study evolution on a molecular scale. I am initially focussing on two epsilon-proteobacterial proteins, PflA and PflB, building on previous work from our lab. PflAB form periplasmic disk structures, thought to function as scaffolding by interacting with other motor components, recruited by an ancestor of epsilon-proteobacteria.
Where did additional proteins, such as PflAB, come from and how did they initially associate with the motor? What changes enabled them to be integral to the motor? What is the order of incorporation of additional components? My approach to tackle these questions is threefold.
Firstly, in situ cryo electron-tomography and subtomogram averaging will yield low-to-intermediate resolution contextual information. Imaging bacterial mutants with a deleted protein of interest helps determine its location within the motor. Earlier work by collaborators identified two proteins, essential for C. jejuni motility. To help understand their functions and determine if they are part of the periplasmic scaffold, I will image their deletion mutants. Imaging other relevant bacteria will help build the motors evolutionary history. Bdellovibrio bacteriovorus is a related bacterium with a potential distant PflA homolog. Determinig the motor structure of this deletion mutant will help establish if the protein is a PflA homolog, and whether the motor is descended from an early precursor before incorporation of other accessory proteins.
Secondly, in vitro work will provide higher resolution information on protein domains and atomic structure. To become part of a complex, a protein must evolve a binding interface. To define protein binding interfaces between periplasmic flagellar proteins (e.g. PflAB, MotB), I will perform pull-down assays for protein pairs, believed to interact, using different truncations to identify interacting regions. Additionally, I will attempt to obtain atomic structures of purified proteins and protein complexes by combination of X-ray crystallography and single particle analysis.
Finally, in silico phylogenetics and sequence analysis will complement wet lab research. To build an evolutionary history of epsilon-proteobacterial motors and help determine order of recruitment of peripheral proteins, we will build a phylogeny of accessory proteins and compare with phylogeny of core flagellar components. PflAB contain many tandem TPR motifs, repeats often present in protein-binding interfaces. We will tpredict PflAB protein-binding regions, comparing sequences between motifs and from dn/ds ratios (rates of non-synonymous and synonymous mutations), which will enhance our understanding of the functional mechanisms of PflAB in the motor.
These sets of structural, bioinformatic, phylogenetic, and molecular biological experiments will be highly informative about the role of these proteins in the flagellar motor, how they perform these functions, their evolutionary mechanisms and path to incorporation, contributing to our
Organisations
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M011178/1 | 01/10/2015 | 25/02/2025 | |||
| 2131359 | Studentship | BB/M011178/1 | 29/09/2018 | 18/01/2023 |