Doctoral Dissertations

Date of Award

5-2023

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Chemistry

Major Professor

Brian K. Long

Committee Members

Brian K. Long, Michael D. Best, Bin Zhao, Barry D. Bruce

Abstract

Integral membrane proteins are prolific targets for the design, development, and delivery of pharmaceuticals. In fact, over 60% of all currently available drugs target these proteins to accomplish their therapeutic effect. However, integral membrane proteins remain the least characterized class of all proteins, accounting for only ~2% of all solved protein structures. One of the primary reasons for this low number of solved protein structures is that many membrane proteins lose their native conformation when extracted using conventional methods (e.g. detergents), convoluting accurate structure determination. In contrast, amphiphilic styrene-maleic acid copolymers (SMAs) were recently discovered to readily isolate membrane proteins via the formation of stable “nanodiscs” that retain an annulus of native lipids. These nanodiscs, commonly referred to as styrene-maleic acid lipid particles (SMALPs), have been shown to retain the proteins native conformation and facilitate precise structural characterization.

Following this discovery, researchers quickly discovered that various other amphiphilic copolymers and related SMA analogues also exhibit the ability to aid in the extraction of membrane proteins. The studies have shown that various polymer characteristics (e.g. molecular weight, monomer incorporation ratio, subunit identity, etc.) play a crucial role in this process, seemingly dictating many aspects of the protein extraction process, such as extraction efficiency, protein selectivity, etc. Herein, I present my contributions to the world’s understanding of amphiphilic copolymer-facilitated protein extraction and expound upon our efforts to promote the efficient and selective extraction of target membrane proteins in their native, or native-like, state.

My first project (Chapter II) explores the modification of a commercial styrene-maleic anhydride copolymer with alkoxyethoxylate sidechains leading to both the selectivity and efficiency of protein extraction being improved. We found that specific sidechains improved both the selectivity and efficiency of the extraction of chlorophyll-containing pigment protein complexes from Thermosynechococcus elongatus. Chapter III focused on the proper storage and preparation of these functionalized polymers to minimize degradation and subsequent protein extraction inconsistencies. The final project discussed (Chapter IV) explores the development of a highly tailorable class of materials that are not susceptible to saponification and retain the ability to efficiently extract trimeric photosystem I from Thermosynechococcus elongatus.

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