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Title: Insights into protein-lipid interactions by structural mass spectrometry
Author: Pyle, Euan
ISNI:       0000 0004 9350 0564
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2020
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Interactions with membrane lipids have long been known to affect a wide range of membrane protein properties including folding, stability, and function. Our understanding of the precise molecular basis of these interactions is currently limited. Here, we combine mass spectrometry (MS)-based techniques with molecular dynamics (MD) simulations, in vivo biochemical assays, and other biophysical techniques to characterise how protein-lipid interactions affect the oligomerisation, conformation, and function of the structurally related xanthine/uric acid transporter UapA from Aspergillus nidulans and the boron transporter BOR1 from Saccharomyces cerevisiae (ScBOR1p). Using native MS and lipidomics, we found that UapA requires phosphatidylethanolamine or phosphatidylinositol binding to form the physiological dimer. A putative lipid binding site at the dimer interface was identified using MD simulations. Lipid binding at this site is essential for formation of functional UapA dimers. Similar analysis of ScBOR1p revealed that this protein is primarily monomeric in detergent-based solution and that phosphatidylethanolamine or phosphatidylserine binding is also essential for its dimerization. Mutation of a putative lipid binding site in ScBOR1p results in loss of lipid-induced dimer formation but has no effect on transport function. Thus, the lipids play slightly different roles in UapA and ScBOR1p. Additional research probed the conformational dynamics of UapA in different lipid compositions using hydrogen-deuterium exchange MS (HDX-MS). The results revealed that protein-lipid interactions stabilise the mobile domain of UapA responsible for substrate transport. HDX-MS also revealed how substrate binding stabilises the inward facing conformation of UapA and that specific mutations stabilise the outward facing conformation of UapA. Together, this work represents the first detailed analyses of the interactions between eukaryotic membrane transporters and their associated lipids.
Supervisor: Byrne, Bernadette ; Hanyaloglu, Aylin Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral