Date of Award
6-26-2026
Date Published
July 2026
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biomedical and Chemical Engineering
Advisor(s)
Liviu Movileanu
Abstract
Protein–protein interactions (PPIs) regulate signal transduction, molecular recognition, and the formation of higher-order biomolecular assemblies. However, quantitative characterization remains challenging in several biologically important contexts, including membrane-associated systems that often require lipid reconstitution, receptor–ligand interactions with complex kinetic behavior, and protein networks that undergo liquid–liquid phase separation (LLPS). This dissertation addresses these challenges by integrating complementary biophysical, biochemical, and imaging approaches to elucidate interaction mechanisms across three representative systems. First, a membraneless experimental platform was developed to interrogate membrane protein interactions in real time without lipid bilayer reconstruction. By coupling engineered with label-free optical biosensing, this strategy direct monitoring of association and dissociation processes while reducing experimental complexity. Second, the binding dynamics of various epidermal growth factor receptor ectodomain (EGFR ECD) isoforms with high-affinity growth factor (GF) ligands, including epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and heparin-binding EGF-like growth factor (HB-EGF), were systematically characterized using biolayer interferometry and surface plasmon resonance. Across ligand–receptor pairs, kinetic analyses revealed reproducible fast and slow dissociation phases, consistent with the coexistence of two binding substates, indicating this is a common feature of GF-EGFR ECD interaction. Our approach could be used for other ligand-receptor systems to directly assess how specific posttranslational modifications influence their interactions. Third, the molecular mechanism by which site-specific protein self-association promotes LLPS was investigated in the chromatin-associated hub protein WD repeat-containing protein 5 (WDR5). Combining biochemical characterization, fluorescence microscopy, and computational analysis identified an defined interaction mode that nucleates WDR5 self-association and drives condensate formation. This demonstrates how discrete contacts within a protein scaffold regulate phase behavior and higher-order assembly. Collectively, these studies advanced our quantitative understanding of how proteins bind, dissociate, and assemble across diverse interaction regimes. This work highlights the value of integrating label-free kinetic biosensing with molecular and imaging measurements to clarify mechanistic principles underlying dynamic protein interaction networks and their roles in cellular regulation and disease.
Access
Open Access
Recommended Citation
Wang, Yazheng, "Quantitative Characterization of Protein-Protein Interactions and Biomolecular Assembly Using Optical and Fluorescence Approaches" (2026). Dissertations - ALL. 2323.
https://surface.syr.edu/etd/2323
