Description
Despite constituting nearly a quarter of the existing proteome, membrane proteins remain some of the most challenging biological molecules to isolate for biochemical and structural characterization. Insights into membrane protein structure and function can aid in understanding of fundamental cellular processes, complex signaling cascades, and the development and progression of disease at a molecular level. This thesis focuses on optimizing strategies for the expression, purification, and characterization of two membrane protein complexes: a bacterial ATP synthase linked to photosynthesis and a neurotrophin receptor complex implicated in neurodegeneration. The multi-subunit ATP synthase complex plays a pivotal role in energy generation and consumption across both autotrophic and heterotrophic organisms. The project described in this thesis aims to characterize this protein complex from a primitive photosynthetic bacterium, Chlorobaculum tepidum, to understand how the complex collaborates with the organism’s photosynthetic system to maintain normal cellular physiology. To isolate the complex, procedures, including detergent usage and sucrose density gradient ultracentrifugation, were optimized to maximize protein stability throughout the membrane extraction process. This work will demonstrate characteristics of the membrane extraction and future projections for its structural determination. The second part of this thesis focuses on the sortilin-p75NTR-proNGF complex, which dictates critical physiological processes in the nervous system and remains an elusive yet prominent contributor to neuronal apoptosis, potentially a leading cause of neurodegeneration. The individual components of the complex could be abundantly expressed and purified by use of the baculovirus expression system in Sf9 insect cells, and preliminary characterizations with electron microscopy indicate the possible formation of the intact complex. Although they play significantly different biological roles, both complexes require specialized techniques to maintain their structural integrity throughout experimental procedures, and together, they illustrate the extreme structural and functional variety that within the membrane proteome.
Details
Contributors
- Kitchel, Heidi (Author)
- Chiu, Po-Lin (Thesis advisor)
- Mazor, Yuval (Committee member)
- Mills, Jeremy (Committee member)
- Arizona State University (Publisher)
Date Created
The date the item was original created (prior to any relationship with the ASU Digital Repositories.)
2024
Topical Subject
Resource Type
Language
- eng
Note
- Partial requirement for: M.S., Arizona State University, 2024
- Field of study: Biochemistry
Additional Information
English
Extent
- 108 pages