During my undergraduate degree, I have worked with the ABCB1 protein (ATP-binding cassette) in Prof Linton’s lab where site-directed mutagenesis was used to explore its effect on transport activity. Two acidic residues were mutated to alanine resulting in a four-day experiment that involved a 5-hour live-cell (HEK 293T), drug transport assay on an LSRII flow cytometer. During my master’s in Prof Mullineaux’s and Dr Darbari’s lab, I worked with two MCE (mammalian cell entry) proteins in Synechocystis PCC 6803 where I created gene knockouts and assessed the effect in chlorophyll via confocal microscopy, ultrastructure analysis and structured illumination microscopy (super-resolution). My previous experience made me realise the passion I had for structural biology and ABC transporters.
ABC transporters couple ATP hydrolysis to the movement of diverse substrates across membranes and are found in all organisms and all membranes. Peroxisomal ABC transporters transport substrates for the beta-oxidation pathway from the cytosol into the peroxisome. Genetic studies show they are of profound importance for the growth and development of the organism. In plants, a single broad specificity transporter accepts a diverse range of acyl-CoA substrates and cleaves them upon transport (a unique activity of this ABC transporter). Previous work from the group has established robust expression, purification protocols and functional for the peroxisomal ABC transporter from Arabidopsis thaliana. I hope to purify the wild type and two mutant forms of AtABCD1 which are predicted to correspond to apo and ATP bound pre hydrolysis states and determine the structure by cryoEM in the presence of different substrates/inhibitors.