Date of Award

Spring 2021

Document Type


Degree Name

Doctor of Philosophy (PhD)


Chemistry & Biochemistry



Committee Director

Steven M. Pascal

Committee Member

Lesley H. Greene

Committee Member

Alvin A. Holder

Committee Member

Erin B. Purcell

Committee Member

Harold C. Riethman


Prostate apoptosis response-4 (Par-4) is an apoptosis-inducing tumor suppressor protein. Full-length Par-4 has previously been shown to be a predominantly intrinsically disordered protein (IDP) under neutral conditions, with significant regular secondary structure evident only within the C-terminal coiled coil domain. However, IDPs can gain ordered structure through the process of induced folding, which often occurs under non-neutral conditions. Previous work has shown that the Par-4 leucine zipper, which is a subset of the C-terminal coiled coil domain, is disordered under neutral conditions, but forms a dimeric coiled coil at acidic pH. Increase in ionic strength was also shown to increase leucine zipper formation. Building on this work, we undertook to study the effects of environment on a naturally occurring Par-4 segment, the cl-Par-4 fragment. During apoptosis, intracellular full-length Par-4 is cleaved at aspartic acid 131 by caspase-3, generating a 24 kilodalton fragment (cl-Par-4). Cl-Par-4 enters the nucleus and inhibits pro-survival genes, thereby preventing cancer cell proliferation.

Here, the structure of cl-Par-4 was investigated using circular dichroism (CD) spectroscopy, dynamic light scattering (DLS), intrinsic tyrosine fluorescence, and size exclusion chromatography with multi-angle light scattering (SEC-MALS). Biophysical characterization showed that under conditions of low salt and neutral pH, cl-Par-4 forms large soluble aggregates. We have clearly identified two disparate conditions under which cl-Par-4 forms non-aggregated largely helical structures. First, with low salt and acidic pH, c l-Par-4 folds into a predominantly alpha helical and coiled coil structure. Second, at neutral pH and high ionic strength, cl-Par-4 forms highly helical tetramers. Together, these results suggest that the cellular environment influences the in vivo structure and self-association state of cl-Par-4 and that the tetramer may be the active conformation under specific intracellular conditions.

A third area of research involves the chemotherapeutic drug cisplatin and its trans isomer transplatin. We have shown that both bind directly to full-length Par-4 and the caspase-cleaved fragment. It appears that this binding interaction occurs through coordination of platinum to sulfur ligands in the protein, such as methionine and/or cysteine residues. This direct binding of cisplatin and transplatin could also subsequently influence apoptotic activity and Par-4 structure.


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