Date of Award

Summer 8-2026

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Steven M. Pascal

Committee Member

Erin B. Purcell

Committee Member

Lisa Shollenberger

Committee Member

Emilia L. Oleszak

Abstract

Non-communicable diseases, including cancer and neurodegenerative disorders, and communicable diseases caused by viral pathogens remain major global health challenges. Despite their diverse etiologies, these diseases are fundamentally governed by the structure, dynamics, and interactions of proteins and RNAs. This dissertation applies structural biology approaches to investigate the tumor suppressor protein Prostate Apoptosis Response-4 (Par-4) and Stem Loop B (SLB) of the Coxsackievirus B3 (CVB3) 3′ cloverleaf RNA to define structural mechanisms underlying their biological functions.

The first project investigated the molecular basis for the unusual requirement of caspase-cleaved Par-4 (cl-Par-4) for high-salt or low-pH conditions to maintain its solubility, stability, and predominantly helical conformation. Based on the hypothesis that electrostatic repulsion within the leucine zipper contributes to this behavior, a D313K mutant was generated to replace a repulsive interaction with a stabilizing salt bridge. Circular dichroism spectroscopy, dynamic light scattering, and NMR spectroscopy demonstrated that the D313K mutation markedly reduced salt sensitivity while preserving the overall tertiary structure, identifying inter-helical electrostatic repulsion as a key determinant of cl-Par-4 stability.

The second project characterized the structure of SLB within the CVB3 3′ cloverleaf, a conserved RNA element implicated in viral RNA replication. NMR spectroscopy revealed that SLB adopts a stable A-form helical stem and a conformationally dynamic loop, suggesting that loop flexibility may facilitate interactions with viral and host factors required for replication complex assembly.

The third project established a preliminary cellular framework for investigating the biological relevance of cl-Par-4 condensate-like phase separation. Fluorescence microscopy and flow cytometry were used to evaluate the localization and apoptotic activity of GFP-tagged full-length and cl-Par-4 constructs in cancer cells, providing an experimental foundation for future studies examining the relationship between Par-4 phase behavior, intracellular localization, and apoptosis.

Collectively, this dissertation integrates three complementary projects unified by a structural biology approach to elucidate how macromolecular structure, electrostatics, and conformational dynamics regulate biological function in both non-communicable and communicable diseases.

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DOI

10.25777/h5nc-v879

ISBN

9780438900288

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Available for download on Thursday, October 12, 2028

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