Doctoral Dissertations
Date of Award
12-2025
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Major
Biochemistry and Cellular and Molecular Biology
Major Professor
Francisco N. Barrera
Committee Members
Rajan Lamichhane, Elizabeth Fozo, Rachel McCord, Amit Joshi
Abstract
Interactions between membrane proteins and their surrounding environment are essential to the maintenance, survival, and modulation of cellular behavior. Receptor tyrosine kinases are membrane proteins that undergo activation through protein-protein oligomerization that modulates these behaviors highlighting the importance of understanding the formation and function of these complexes. Thus, the development of techniques that allow us to dive deeper into these interactions will provide crucial insight into the inner workings of cellular networks. In this work, we present “SiMPull-POP,” a single-molecule pulldown-photobleaching method that utilizes a co-polymer compared to traditional detergent methodologies to solubilize membrane proteins within nanoparticles that contain endogenous lipids (Chapter II). This co-capturing allows for the study of proteins in a more native-like environment. In the work presented here, we validated SiMPull-POP using a known inducible-dimer system and then applied this method to study the self-assembly of the RTK EphA2 in response to ligand stimulation and membrane modification. Next, we investigated the ROR RTK family where the mechanism and modulation of their oligomerization is still not well understood. This RTK family is comprised of ROR1 and ROR2 that are key regulators of embryogenesis, aiding in the development of bone, muscle, and neural tissues. To provide insight into the assembly mechanism of this RTK family we quantified the self-assembly of ROR2 (Chapter III) and ROR1 (Chapter II) using SiMPull-POP. Elucidating that the self-assembly of ROR1 is promoted by cholesterol in the membrane while ROR2 self-assembly is modulated by calcium. In conclusion, the work presented here contributes a quantitative method to monitor protein interactions that has both confirmed and provided new insights into the self-assembly of membrane proteins.
Recommended Citation
Ward, Alyssa E., "Receptor Tyrosine Kinase-like Orphan Receptors: Elucidating receptor self-assembly in a native-like membrane environment at single molecule resolution. " PhD diss., University of Tennessee, 2025.
https://trace.tennessee.edu/utk_graddiss/13648
Included in
Biochemistry Commons, Cancer Biology Commons, Other Biochemistry, Biophysics, and Structural Biology Commons