understanding the fundamental principles behind the influences of native and non-native entanglements on protein structure, function, and misfolding
Open Access
- Author:
- Sitarik, Ian
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 12, 2025
- Committee Members:
- William Noid, Major Field Member
Arthur Lesk, Outside Unit & Field Member
Mark Hedglin, Major Field Member
Edward O'Brien, Chair & Dissertation Advisor
Kenneth Knappenberger, Program Head/Chair - Keywords:
- Protein folding
protein entanglements
protein misfolding
synonymous mutations
MD simulations
proteomics
non-covalent lasso entanglements - Abstract:
- Protein folding and misfolding are fundamental processes influencing cellular function, proteostasis, and evolution. This thesis investigates the dynamics of protein misfolding, focusing on soluble, long-lived misfolded states and their implications for function and proteostasis. Using computational simulations, statistical modeling, structural analyses, and mass spectrometry, I identify mechanisms through which misfolded proteins bypass chaperone systems and persist in cells. My results predict that approximately one-third of all proteins in E. coli can adopt misfolded conformations that remain soluble but are less functional, with some persisting for months. These misfolded states often exhibit native-like surface properties yet are kinetically trapped due to changes in self-entanglement that will require significant unfolding of the protein to correct. A careful analysis of the currently known structures in the protein data bank show significant portions of the proteomes across different organisms contain these novel self-entanglement motifs called non-covalent lassos and that certain biological and molecular processes are enriched in them. A high-throughput analysis of mass spec data probing structural changes in the E. coli proteome upon refolding reveals that proteins with native entanglements are more prone to misfolding, with chaperone systems selectively rescuing this misfolding in essential proteins. These findings elucidate a failure-to-form mechanism of misfolding and suggest evolutionary pressures shape loop stability that are dependent on protein essentiality. Together, this work provides a comprehensive framework for understanding a novel kinetic trapping protein misfolding mechanism, their functional consequences, and their interplay with cellular quality control systems, offering insights into their evolutionary and physiological significance.