Remarkable in vitro Assemblies of a Bacterial Cytoskeletal Protein, FilP and its Derivatives

Alcock, Emily (2020) Remarkable in vitro Assemblies of a Bacterial Cytoskeletal Protein, FilP and its Derivatives. Doctoral thesis, University of East Anglia.

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Intermediate filaments form a part of the cytoskeleton in eukaryotes. Intermediate filaments can self-assemble into large, non-polar higher order structures and have a characteristic domain architecture. Few homologues of intermediate filaments in bacteria have been identified. The protein FilP from Streptomyces coelicolor is thought to be intermediate filament-like and is the focus of this thesis.
In this thesis FilP is compared to eukaryote intermediate filaments to show that FilP does have a great deal of similarity to those and fits the classical definition of intermediate filaments in many ways, although there are a few key differences. A notable difference is the unusual 51-mer repeat sequence seen in its coil 2. Characterisation of FilP higher order assembly in vitro reveals the higher order structures formed in varying buffer conditions using transmission electron microscopy. FilP was found to form a rope structure, characteristic of intermediate filaments, and other structures including a striated structure.
A series of FilP variants were generated, expressed and purified which were designed to identify the essential regions for FilP for higher order assembly. The variants were observed by transmission electron microscopy which that the C terminal end of the coil 2 subdomain was essential for the formation of higher order structures. Expression of these FilP variants in Escherichia coli yielded surprising results in that the length of the cells when grown on cellophane increased dramatically from a normal length. Intriguingly, cells expressing a small section of 51-mer repeat sequence dramatically affected the morphology of the cells. This suggests that FilP interacts with the cell division machinery within Escherichia coli. A FilP knockout strain of Streptomyces coelicolor revealed that FilP is involved in the structural integrity of the cell. FilP localisation within Streptomyces coelicolor was also observed.
These experiments clearly show that FilP is involved in the cytoskeleton of the cell and displays characteristics of intermediate proteins. However, it does not exactly fit the strict definition of intermediate filaments. This research opens up the discussion what constitutes an intermediate filament.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Nicola Veasy
Date Deposited: 06 Apr 2022 10:40
Last Modified: 30 Nov 2023 01:38

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