Date Approved

4-15-2026

Embargo Period

4-15-2026

Document Type

Dissertation

Degree Name

Ph.D. Cell and Molecular Biology

Department

Cell and Molecular Biology

College

Rowan-Virtua School of Translational Biomedical Engineering & Sciences

Advisor

Brian P. Weiser, Ph.D.

Committee Member 1

James Holaska, Ph.D.

Committee Member 2

Zhiwei Liu, Ph.D.

Committee Member 3

Natalia Scherbik, Ph.D.

Committee Member 4

Sergei Borukhov, Ph.D.

Disciplines

Biochemistry, Biophysics, and Structural Biology | Life Sciences | Molecular Biology

Abstract

Proteins are dynamic molecules whose functional landscapes extend beyond static structural representations. Transient cryptic pockets (buried cavities that emerge through protein motion) represent underexplored targets for allosteric regulation and drug discovery. General anesthetics are small, lipophilic, weakly polar molecules that are well suited to partition into such hydrophobic sites. In this work, environment-sensitive fluorescence, photoaffinity labeling, mass spectrometry, mutational analysis, and microsecond molecular dynamics simulations were integrated to discover and characterize a cryptic, highly apolar binding site within the hydrophobic core of human proliferating cell nuclear antigen (PCNA), a central scaffold in DNA replication and repair. Using the solvatochromic fluorescent anesthetic analog 1-aminoanthracene (AMA) and a diazirine-containing propofol analog (AziPm), the principal interaction site was localized to residues L90 and L101. Substitutions at these positions altered pocket architecture and AMA fluorescence spectra, confirming their structural and chemical importance. Clinically-used anesthetics, including propofol and sevoflurane, compete with AMA for the site. MD simulations revealed that pocket formation occurs transiently in wild-type PCNA (~25% open) and with enhanced probability in the L90I variant (~75% open). Ligand occupancy thermodynamically stabilizes the PCNA trimer and reduces subunit exchange, demonstrating that occupation of this buried site is functionally coupled to clamp dynamics. Together, these results establish PCNA as a clamp that harbors a functionally coupled, druggable cryptic site and provide a general framework for discovering and exploiting buried allosteric pockets in proteins.

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