Dissertations, Theses, and Capstone Projects

Date of Degree

6-2025

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy

Program

Biochemistry

Advisor

Sebastien Poget

Advisor

Sharon Loverde

Committee Members

Rupal Gupta

Ruth Stark

Yael David

Michael Brenowitz

Subject Categories

Biochemistry | Biophysics | Structural Biology

Keywords

S100A12, Nucleosome Core Particle, Histone, Calcium Binding, Oncogenic Mutations

Abstract

Calcium signaling and chromatin remodeling are fundamental regulatory processes that control protein activity and gene expression, respectively. This thesis investigates the molecular mechanisms by which local ligand binding and structural perturbations propagate allosteric effects across larger protein systems, using two distinct yet mechanistically analogous models: the calcium binding protein S100A12 and the nucleosome core particle. These studies are unified by a shared emphasis on how physical forces govern biological function.

In the first part, we explore calcium mediated allostery in S100A12, focusing on the distinct roles of its two EF-hand calcium binding loops (EF-I and EF-II). Through a combination of isothermal titration calorimetry, NMR spectroscopy, and mutational analysis, we find that EF-II acts as the primary driver of the conformational switch upon calcium binding. However, EF-I, though dispensable for the core structural transition, plays a critical modulatory role in stabilizing the dynamic receptor-binding interface. Notably, the E31A mutation, which eliminates EF-I calcium binding, leads to mildly diminished calcium affinity in EF-II and changes in the flexibility of the C-terminal tail, suggesting a form of structural cooperativity. These results define EF-I as an enhancer of conformational fidelity and receptor readiness, providing new insight into the activation mechanism of S100 proteins.

The second part investigates how oncogenic mutations affect the physical stability and remodeling behavior of the nucleosome core particle. We focus on two cancer associated histone mutations: H2B E76K and H4 R92T. Using differential scanning calorimetry, thermal stability assays, and molecular dynamics simulations (provided by our collaborators), we reveal the molecular mechanism by which E76K enhances dimer exchange while inhibiting nucleosome sliding, and R92T promotes sliding but reduces exchange. These divergent mechanisms destabilize chromatin organization in distinct ways: E76K likely leads to enhancer hyperpriming and transcriptional noise, while R92T likely enables excessive chromatin mobility and epigenetic instability. Our analysis redefines key thermodynamic transitions in the NCP dissociation pathway and provides a unified model for how histone mutations disrupt chromatin compaction, accessibility, and ultimately, cell fate.

Together, these studies advance our understanding of how local perturbations, whether calcium binding or point mutations, can reshape global protein structure, dynamics, and function. By integrating biophysical measurements with structural and computational analysis, this work offers mechanistic insight into protein allostery and chromatin regulation, with implications for inflammation, signal transduction, and cancer biology.

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