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Polymer-Based Nanodiscs for Structural and Functional Analyses of Bacterial and Mammalian Membrane Proteins
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- Author / Creator
- Mansoore Esmaili
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Unlike cytosolic proteins, membrane proteins (MPs) are embedded within the plasma membrane and the lipid bilayer of intracellular organelles. MPs serve in various cellular processes such as ion and metabolite transports, bioenergetic processes, signal transductions, and cell-cell communications. Making up nearly 25% of the human proteome, MPs account for over 65% of the current drug targets.
The structural integrity and functional dynamics of MPs have been evolved in the lipophilic membrane bilayers. However, the reconstitution and in vitro characterization of MPs in their native lipid bilayers remain challenging.
Since the revelation of the first structure of a membrane protein in the 1970s, small molecule detergents have been used for the purification of MPs from their lipid bilayer into lipid-like artificial assemblies of detergent micelles. However, detergents remove the native lipid molecules away and, consequently, compromise the activity and stability of MPs. As such, lipid-dependent conformational studies of MPs and structural analysis of membrane-embedded enzymes with lipid substrates are incredibly demanding. The development of other membrane mimetic systems (such as bicelles, short synthetic polymers or amphipols, and protein-based discoidal nanodiscs) has facilitated the accommodation of synthetic lipids to stabilize MPs. Yet, the preparation of these membrane mimetics still relies on the use of detergents.
Synthetic amphipathic polymers present an invaluable tool for truly detergent-free excision and liberation of superstructures of MPs and their surrounding annular membrane bilayer in donut-shaped nanoparticles or discs. Among all, styrene-co-maleic acid polymers (SMAs) are the most well-studied amphipathic polymers with the highest yield for purification of MPs. Some drawbacks such as sensitivity to acidic pH and high concentration of divalent cations, interference with the spectroscopic analysis of proteins, high polydispersity index (PDI) and nonspecific interaction with protein surfaces have hindered the ultimate optimal utilization of SMA polymers for structural characterization of MPs in membrane bilayer and hence for drug discovery purposes.
In this thesis, I discuss synthesis, chemical modifications, and biophysical characterization of multiple series of novel non-RAFT amphipathic polymers that demonstrate improved behaviors at acidic pH and high concentration of calcium. Some of these unique amphipathic polymers (such as methyl stilbene-alt-maleic acid copolymers) show very low polydispersity (PDI ~1) and strict alternation in sequence (a well-defined sequence of co-monomers), and some exhibit distinct spectrophotometric profiles. Furthermore, I address the application of these novel polymeric detergents for the purification and functional analysis of a lipid A palmitoyl transferase, PagP, from the outer membrane of E. coli, as well as for detergent-free purification and lipid analysis of the infectious mammalian prion protein (PrPSc) directly from the brains of infected rodents. -
- Subjects / Keywords
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- Graduation date
- Spring 2020
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- Type of Item
- Thesis
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- Degree
- Doctor of Philosophy
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- 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.