Date of Award

Fall 2025

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Krisztina Varga

Second Advisor

Xuanmao Chen

Third Advisor

Victoria Jeffers

Abstract

Proteins are nature’s dynamic molecular machines, operating across a spectrum from highly ordered scaffolds to intrinsically disordered systems, and executing functions that depend on both precise structures and conformational flexibility. Yet, the mechanistic rules linking sequence, structure, and activity particularly in disordered proteins and specialized functional adaptations remain incompletely understood. This dissertation addresses that gap by integrating structural biology, biophysics, and protein engineering to dissect molecular recognition, stability, and function across three diverse yet thematically connected protein systems. Building on this understanding of protein structure and function, the following four projects apply these concepts to address diverse biological questions, ranging from protein interactions and structural requirements to proteins’ roles in adaptations to extreme environments, protein stability and evolution, and strategies for inhibiting viral proteins. Chapter 1 will provide a short introduction to Nuclear Magnetic Resonance (NMR) structural methods utilized in this work. In the first project (Chapters 2), I investigate the bacterial hub protein PopZ from Caulobacter crescentus, an intrinsically disordered polar organizing scaffold that anchors chromosome origins, recruits regulatory factors, and coordinates asymmetric cell division by gathering proteins at the poles. Its 11 known binding partners vary greatly in sequence and structure, raising the central question: how can PopZ bind to such a wide variety of proteins while remaining highly selective? In this context, PopZ’s function is determined not only by its own structure but also by the structures of its diverse binding partners. I have utilized Nuclear Magnetic Resonance (NMR) spectroscopy to characterize the interaction between a truncated version of wild-type PopZ and one of its client proteins, CpdR. I also examined how mutant PopZ proteins interact with CpdR to gain insight into the binding interface and determined the preliminary structure of one mutant PopZ protein. These findings reveal how subtle adjustments in helix can tune affinity and specificity in bacterial hub proteins. Extending this work, I began examining the interaction from the perspective of the client proteins, taking the first steps toward understanding how such structurally diverse proteins recognize PopZ. These studies lay the groundwork for uncovering how PopZ can exhibit the dual properties of binding a wide variety of proteins while maintaining high specificity. The second project focuses on a hyperactive insect antifreeze protein, ApAFP752, from the beetle Anatolica polita (Chapter 3). In collaboration with other lab members, using solution NMR spectroscopy alongside ice recrystallization inhibition (IRI) and thermal hysteresis (TH) assays, we determined that ApAFP752 adopts a β-helical architecture with a threonine-rich ice-binding surface including regularly spaced T-X-T motifs. Structural analysis and comparative modeling with other insect AFPs indicate that surface regularity and side-chain arrangement are critical for its high IRI activity. Guided by these insights, I rationally redesigned the ice binding surface to improve motif spacing and packing. The engineered variant achieves approximately a fivefold improvement in IRI efficiency, demonstrating a direct, structure-guided enhancement of cryoprotective function. These results provide insights into the determinants of AFP hyperactivity and suggest potential strategies for engineering biomolecules with improved ice control capabilities for applications in cryopreservation, agriculture, and food science. In the third line of research, I test the misfold avoidance hypothesis by directly measuring thermodynamic stability across a panel of Bacillus subtilis proteins spanning a wide range of native expression levels (Chapter 4). Using circular dichroism spectroscopy, I find that stability is only weakly correlated with expression, challenging the universality of the proposed link between high expression and increased folding robustness. This work adds experimental nuance to evolutionary models of protein stability. Finally, I explore the potential of repurposing thiadiazolidinone (TZDZ) derivatives as antiviral agents against SARS-CoV-2 by targeting its main protease (MPro). Through kinetic analysis, I confirm that the most promising candidate, CCG-50014, acts as a covalent inhibitor, with an inhibitory profile consistent with irreversible active-site modification (Chapter 4). This finding provides a foundation for further optimization of TZDZ derivatives as antiviral therapeutics. Collectively, these projects illuminate the unifying theme that both rigid structural motifs and dynamic disorder can be finely tuned to optimize function, whether in mediating selective yet adaptable protein interactions, enhancing extreme-environment adaptations, or shaping stability and inhibition profiles. By combining detailed structural and dynamic characterization with rational design, this work advances our understanding of structure–function relationships and demonstrates how such insights can be translated into practical innovations in biotechnology, evolutionary biology, and therapeutic development.

Download

Share

COinS