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Title: Frontiers in protein-lipid interactions studied by native mass spectrometry
Author: Hoi, Kin Kuan
ISNI:       0000 0004 7653 0104
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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Coordination between membrane proteins and their surrounding lipid environment dictates the function of biological membranes. While the study of membrane protein-lipid interactions is challenging for well-established techniques due to their transient and dynamic nature, recent developments in native mass spectrometry (MS) enables interrogation of membrane protein-lipid interactions in a label-free and high-resolution manner. This thesis presents two major developments in the study of membrane protein-lipid interactions via native MS: (1) the development of suitable MS methods for the transfer and characterization of nanodisc assemblies, promising membrane mimetics alternative to classical detergent micelles; and (2) the characterization of the lipid interactions with class A GPCRs. Nanodiscs were transmitted into the gas-phase and collisionally activated to produce mass spectra, which were highly complex. To interpret these spectra, a dual Fourier transform method (Chapter 2) and a mass defect analysis (Chapter 3) were developed, allowing the determination of the lipid composition and the stoichiometries of the lipid/protein components of the nanodiscs. Analysis of the spectral results revealed that nanodisc-based MS provides novel insights into annular lipid interactions. In addition, the dissociation behavior of nanodiscs upon collisional activation was explored to understand how best to characterize nanodiscs in the gas-phase. In the second part of this thesis, the lipid interactions of class A G protein-coupled receptors (GPCRs) were carefully characterized by native MS and coarse-grained molecular dynamics simulations. Class A GPCRs were shown to preferentially bind phosphatidylinositol bisphosphate (PIP2) over related lipids and potential PIP2-binding hotspots were identified. The effects of PIP2 on GPCR-G protein coupling were then investigated employing a Gα protein mimetic. Together, this information leads to a hypothesis that PIP2 binding modulates GPCR-G protein coupling and identifies a potential lipid-binding motif. Overall, therefore this thesis develops two novel methodologies, the native MS of nanodiscs and G protein coupling, both of which facilitate studies of protein-lipid interactions in challenging systems.
Supervisor: Robinson, Carol Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available