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Title: Structural studies of the multi-drug resistance protein P-glycoprotein (ABCB1)
Author: Thonghin, Nopnithi
ISNI:       0000 0004 7657 0587
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2018
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P-glycoprotein (P-gp or ABCB1) is a membrane-bound active transporter belonging to the ABC protein superfamily. It is responsible for xenobioIc efflux and also contributes to multidrug resistance in diverse diseases including cancer and epilepsy. P-gp has been increasingly recognised as a potential target for future therapeutics. Although the protein has been studied for decades, understanding of the P-gp transport mechanism is still incomplete. Two P-gp orthologues, mouse (m) and human (h), were therefore expressed in yeasts and purified in the presence of the detergent, n-Dodecyl-β-D- Maltoside (DDM). Purified proteins were examined for aggregation and monodispersity via dynamic light scattering (DLS) and their thermal stability was determined by an assay using a thiol-specific dye (CPM). ATPase activity, measured in a detergent environment, showed that the proteins were active with a basal activity of 60 ± 4 and 35 ± 3 nmol/min/mg for mP-gp and hP-gp, respectively. Crystallisation trials were conducted in the presence of nucleotide. In meso crystallisation using commercial monoolein pre- dispensed plates yielded hexagonal crystal-like objects however they failed to diffract X- rays. P-gp samples were also subjected to cryo-EM where mP-gp in the post-hydrolytic (ADP-bound, vanadate-trapped) state provided the highest resolution dataset that led to a reconstruction of 3D density map at the resolution of 7.9 Å which showed an inward- facing conformation. Rigid-body model fitting unveiled densities that were not accounted for by the fitted model illustrating new features such as bound ADP, extended NBD1- TMD2 linker and alternative allocrite-binding sites. Ultimately, the knowledge of P-gp conformation alteration was enhanced and a refined alternating access mechanism of P- gp was proposed based upon information derived from this study.
Supervisor: Ford, Robert ; Doig, Andrew Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: protein structure ; membrane protein ; P-gp ; cryo-EM ; ABC transporter