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Studying Protein–Glycolipid Interactions and Membrane Peptides and Proteins using Electrospray Ionization Mass Spectrometry and Model Membranes

  • Author / Creator
    Li, Jun
  • Membrane systems, including glycolipids (GLs) and membrane peptides and proteins (MPs), play an important role in many cellular processes, such as singling, cellular recognition, transportation, and energy conversion. However, the amphipathic nature of GLs and MPs makes their analysis challenging. Nanoscale lipoprotein model membranes (MMs) provide a native like lipid environment to solubilize them. This thesis focuses on the development of electrospray ionization mass spectrometry (ESI-MS) based methods combined with lipoprotein MMs for discovery and characterization of GL receptors of glycan-binding proteins (GBPs) and to investigate stoichiometry and conformations of MP complexes.
    Chapter 2 describes the development of the catch-and-release (CaR) ESI-MS assay, combined with picodiscs (complexes comprised of saposin A and lipids, PDs), to screen GL mixtures against water-soluble GBPs to detect specific interactions. The proof-of-concept experiments were performed by screening PDs containing a small library of purified gangliosides against the B subunit homopentamer of cholera toxin (CTB5) and a sub-fragment of toxin A from Clostridium difficile (TcdA-A2), which demonstrated the simultaneous detection of both high and low affinity interactions. Screening mixture of GLs extracted from porcine brain and a human epithelial cell line against CTB5 successfully identified high affinity GL ligands present in both GL mixtures. Finally, a comparison of the present results with data obtained with the CaR-ESI-MS assay implemented using nanodiscs (NDs) revealed that the PDs exhibited similar or superior performance to NDs for GBP–GL binding measurements.
    Chapter 3 reports the first detailed investigation into the composition, heterogeneity and structure of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine-containing PDs (POPC-PDs) in acquous solutions using high resolution ESI-MS, multi-angle laser light scattering (MALLS) and molecular dynamics (MD) simulations. The ESI-MS and MALLS data revealed that the size and composition of POPC-PDs are dependent on pH – predominantly as a SapA dimer at acidic pH; predominantly as a SapA tetramer in freshly prepared solutions at neutral pH and converts to SapA trimer over the course of hours. Comparison of measured collision cross sections (Ω) with values calculated for gaseous ions from modelling suggests that the solution structures are largely preserved in the gas phase, although the lipids do not maintain regular bilayer orientations.
    Chapter 4 describes the use of passively-loaded PDs (PLPDs), prepared by incubating phospholipid PDs with GL or GL mixture (in the form of glycomicelle) in aqueous solution, for CaR-ESI-MS screening of GLs against CTB5 and compares their performance with pre-loaded PDs, prepared directly from a mixture of phospholipid and GL(s). GM1 binding to CTB5 measured for PLPDs prepared from GM1 is indistinguishable from that observed with pre-loaded GM1 PDs. GL binding to CTB5 measured for PLPDs prepared from GLs extracted from pig and mouse brain revealed that the PLPDs allow for the detection of a greater number of ganglioside ligands than pre-loaded PDs. Together, these results suggest PLPDs may have advantages over conventionally prepared PDs for screening GLs against GBPs using CaR-ESI-MS.
    In Chapter 5, the gas-phase conformations of dimers of the channel-forming membrane peptide gramicidin A (GA), produced from NDs, are investigated using ion mobility separation (IMS)-MS and MD simulations. GA dimer is readily transferred from phospholipid NDs to the gas phase by ESI and it suggested that the ion conducting single stranded head-to-head helical conformation of the dimer was preserved in the gas phase. Notably, the conformation of GA dimers produced from NDs was found to be different from those determined directly from organic solvent and phospholipid vesicles, which suggests that the method used to deliver the peptide complexes from the lipid bilayer to the gas phase may influence the conformations of the gaseous ions.

  • Subjects / Keywords
  • Graduation date
    Fall 2018
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3610W76V
  • License
    Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.