Integral membrane proteins represent nearly 25% of protein encoding genes yet account for less than 2 percent of solved structures in protein databases. This underrepresentation is due to the difficulty in membrane protein isolation with the use of detergents. The difficulty in structural characterization following isolation has been alleviated by use of amphipathic polymers that stabilize proteins by maintaining the native protein environment. Of these amphipathic copolymers, SMA2000—a commercially available styrene-maleic anhydride copolymer with a 2:1 styrene to maleic anhydride ratio—has demonstrated broad utility in the isolation and subsequent characterization of a wide range of integral membrane proteins. However, there are some limitations to the use of SMA2000 that may arise from a lack of control of polymer parameters such as sequence, composition, and dispersity. Methyl substituted stilbene and maleic anhydride copolymers with controlled sequences and compositions demonstrated membrane solubilization and protein isolation activity at levels comparable to SMA2000, with several advantages that include an increase in the useable pH range and the size and homogeneity of polymer-lipid particles. Regrettably, the synthesis of methyl substituted stilbene-maleic anhydride copolymers is not without challenges that would make laboratory scale-up difficult. The use of anisole as a polymerization solvent mitigates gelation during poly((E)-4-methylstilbene-alt-maleic anhydride) synthesis. Additionally, unsymmetrical dimethyl-substituted stilbenes and monosubstituted stilbenes ((E)-4-tert-butylstilbene, (E)-4-methoxystilbene, (E)-2-methoxystilbene, methyl (E)-4-carboxylatestilbene, and (E)-4-trifluoromethylstilbene) copolymerize with maleic anhydride without gelation. Rates of conversion reveal that stilbenes with donor substituents copolymerize with maleic anhydride faster than stilbenes with acceptor substituents. The reversible addition–fragmentation transfer (RAFT) controlled radical polymerization technique afforded poly((E)-4-methylstilbene-alt-maleic anhydride) copolymers with controlled sequence, composition, molecular weight, and dispersity. Amphipathic copolymers with fluorescent activity are also desired for membrane protein solubilization. A series of donor-acceptor di-substituted stilbene and maleic anhydride copolymers with fluorescence activity have been synthesized and characterized. These copolymers possess electron-donating and electron accepting substituents on each phenyl ring of the stilbene monomer. These copolymers exhibit red-shifts that vary in position and width with changes in substituent. / Doctor of Philosophy / Proteins embedded in biological membranes—integral membrane proteins—are valuable drug targets due to their significance in a variety of essential cellular functions such as signaling and transport. Structural and functional characterization of integral membrane proteins is necessary to develop drugs that target them. However, despite their importance, integral membrane proteins remain a challenging target for structural characterization. Integral membrane proteins must be removed or solubilized from the lipid bilayer for structural characterization and the typical detergent extraction process often results in protein destabilization or irreversible protein deactivation. Polyanions have emerged as suitable alternatives to the solubilization of integral membrane proteins. Unlike detergents, polyanions solubilize integral membrane proteins with a small portion of the lipid bilayer intact. In this way, the integral membrane protein is removed from the lipid bilayer, but it maintains its structural and functional integrity allowing for structural and functional characterization. Hydrolyzed poly(styrene-co-maleic anhydride) is the most widely used polyanion for integral membrane protein solubilization, however, the sequences and compositions of poly(styrene-co-maleic anhydride) copolymers are not uniform and these inconsistencies have been shown to effect isolated integral membrane protein yield and purity. Hydrolyzed strictly alternating copolymers of substituted-stilbene and maleic anhydride have uniform sequences and compositions and have demonstrated integral membrane protein extraction activity at levels comparable to or greater than the commonly used hydrolyzed poly(styrene-co-maleic anhydride) copolymers. The work described in this dissertation focuses on optimized syntheses and characterization of several substituted-stilbene and maleic anhydride copolymers for the solubilization of integral membrane proteins. The choice of stilbene substituent is shown to affect poly(substituted-stilbene-alt-maleic anhydride) properties such as solubility and fluorescence activity.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/108276 |
| Date | 10 February 2022 |
| Creators | Brown, Chanelle Jasmine |
| Contributors | Chemistry, Gandour, Richard D., Long, Timothy E., Matson, John B., Turner, Sam Richard |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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