Structural analysis of transferred outer membrane protein and bd1904 family of Bdellovibrio bacteriovorus

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Parr, Rebecca (2023). Structural analysis of transferred outer membrane protein and bd1904 family of Bdellovibrio bacteriovorus. University of Birmingham. Ph.D.

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Abstract

Antimicrobial resistance is an ever-increasing global issue and alongside traditional therapies, alternative approaches are needed. One new biological therapy being investigated is the use of \(\textit{Bdellovibrio bacteriovorus}\), a periplasmic predator of fellow Gram-negative bacteria, including multi-drug resistant pathogens. Despite this intriguing lifestyle, much remains unknown about the molecular mechanisms that enable predation. To further our understanding, X-ray crystallography has been employed to determine the structure and function of membrane and periplasmic proteins that are predicted to play essential roles during invasion. Structural biology is an effective tool to investigate proteins of interest as \(\textit{B. bacteriovorus}\) has limited homology to other bacteria.

The primary focus of this thesis is the characterisation of a cryptic outer membrane protein that has previously been shown to transfer from the outer membrane of predator to the inner membrane of prey during predation. The structure was solved using X-ray crystallography of refolded protein from inclusion body material in \(\textit{E. coli}\). Through a large amount of optimisation crystals diffracted to a resolution that could be used to generate a model of the protein, improving the resolution from 10 Å to 2.8 Å. The data collected enabled building of the protein in its entirety, revealing an N-terminally-plugged oval-shaped 16-stranded b-barrel which unlike all other porin structures to date forms a pentamer. The structure and oligomericstate have been validated using cryo-EM of a construct from native membranes. Alongside probing the structure, the porin has been initially characterised using liposome swelling assays, electrophysiology, and the generation of an in-frame deletion in \(\textit{B. bacteriovorus}\).

The secondary focus of this PhD has been to structurally characterise three periplasmic proteins (Bd1645, Bd2126 and Bd2778), which are sequence homologs to Bd1904. bd1904 is the most upregulated gene upon entry into the bdelloplast, but despite previous investigation the protein function is unknown. The crystal structure of Bd2778 shows that it is a structural homolog to Bd1904, which has enabled the development of two working hypotheses for the wider family.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):