I started my PhD at the Astbury Centre for Structural Molecular Biology in 2018 as part of the White Rose BBSRC DTP cohort. I am supervised by Prof Adrian Goldman and Prof Peter Henderson and work on the recently discovered Proteobacterial Antimicrobial Compound Efflux (PACE) family of bacterial multidrug transporters. My PhD will focus on structural biology and biophysical techniques to further characterise these proteins and their transport mechanism.
Before coming to Leeds, I studied for an integrated masters in Biochemistry at the University of York, graduating with first class honours. In my third year I was part of a group project supervised by Prof Tony Wilkinson at York Structural Biology Laboratory (YSBL) where I successfully expressed an ABC family periplasmic substrate-binding protein from the equine pathogen Rhodococcus equi heterologously in Escherichia coli. My first foray into protein biochemistry, however, was the summer before my third year, when I received a Biochemistry Society Summer Vacation Studentship to work in Dr Alison Parkin’s lab at the Department of Chemistry. I helped establish a protein purification protocol for a periplasmic redox protein from Campylobacter jejuni expressed in E. coli in the lab. I developed a redox assay to test the protein’s activity and rationally designed short peptides to investigate the enzyme’s selectivity, using bioinformatics tools and AutoDock Vina to guide my work. I gained experience in protein purification techniques and solid-phase peptide synthesis. I enjoyed the smell of healthy E. coli so much that I took up a second summer studentship, funded by the BBSRC, the following year in Dr Michael Plevin’s lab in the Department of Biology. Here, I further developed my skills in protein purification and biochemistry, optimising a fluorescence-based assay to investigate a DNA helicase. In my fourth year, I switched gears and worked in Dr Caroline Dessent’s lab in the Department of Chemistry on a project entitled ‘Photochemistry of protonated and deprotonated 2-thiouracil’. I used laser photodissociation spectroscopy to investigate the excited states of ionised 2-thiouracils and their decay pathways. I also analysed time-dependent density functional theory calculations on these systems to complement my experimental data.
Pathogens increasingly exhibit resistance to a multitude of synthetic antimicrobial agents and biocides, making infectious diseases a global threat to health. Their ability to do this is based on a number of molecular mechanisms, one of which is the upregulation of multidrug efflux pumps - transport proteins that extrude a wide variety of compounds from cells. The family of proteins I work on, Proteobacterial Antimicrobial Compound Efflux (PACE) family, has so far been studied through an ongoing collaboration of our lab with Dr Karl Hassan (The University of Newcastle, Australia) and Prof Ian Paulsen (Macquarie University, Australia). The PACE prototype, AceI, was first discovered upon chlorhedixine challenge of the Gram-negative pathogen Acinetobacter baumannii and subsequent analysis of the transcriptomic response. AceI was upregulated in response to the biocide and further studies demonstrated that it actively exports chlorhexidine out of the cell. Since this initial discovery, recognition of further biocides has been demonstrated for a number of homologues and there is evidence to support a mechanism of secondary active transport for AceI.
You can listen to Dr Karl Hassan talk to the Naked Scientists podcast about the latest paper on PACE proteins and why this research is important here.
In order to elucidate the fundamental molecular mechanism of PACE proteins, obtaining the structure of one of the homologues is essential and that is the main objective of my project. In addition to structural biology tools and methods, I will use biophysical methods, such as protein reconstitution into liposomes and fluorescence-based transport assays, to further characterise the substrate specificity and transport activity of these proteins.
- MBiochem Biochemistry (1st Class), University of York, 2018