Degree Type

Dissertation

Date of Award

2021

Degree Name

Doctor of Philosophy

Department

Chemistry

Major

Chemistry

First Advisor

Vincenzo Venditti

Abstract

Despite the continuous reports in literature suggesting conformational disorder and structural heterogeneity playing an essential regulatory role in several native enzymes, disentangling the conformational dynamics / structure / function relationship remains a challenging avenue that weighs heavily on the development of innovative biophysical methods capable of providing atomic-level details. Indeed, obtaining an atomic-resolution understanding of protein dynamics, and the processes they are involved in, is essential for engineering effective strategies for therapeutic intervention against human associated disease. NMR methodologies have become a staple in such studies as they offer a way to extract atomistic information of the structure, dynamics, kinetics, and thermodynamics of enzymes under near physiological conditions.Alkbh5 is a member of the AlkB family (AlkB1-8 and FTO), which are non-heme Fe2+ / α-KG-dependent dioxygenases that catalyze the repair of modified nucleobases and amino acids through an oxidative demethylation process. Specifically, Alkbh5 selectively demethylates N6-methyladenosine, an epigenetic mark involved in physiological processes such as stem cell differentiation and carcinogenesis. Previous studies have shown AlkB dioxygenases to be highly dynamic enzymes where the transitions from inactive, disordered, states to active, ordered, conformations are induced through well-orchestrated mechanism of sequential substrate / co-factor binding events. Albeit AlkB enzymes have proven to be of significant importance, accurate characterization of the dynamics and structural models of an AlkB enzyme at each major step in the reaction cycle has not been accomplished. This dissertation focuses on characterizing and understanding the regulatory role played by conformational dynamics in the substrate recognition process of Alkbh5. Specifically, this dissertation includes 1) an introduction to some of the NMR methods implemented for investigating Alkbh5, 2) the protocol developed for expression and purification of mg quantities of Alkbh5 suitable for NMR studies, 3) the NMR backbone resonance assignments of Alkbh5 necessary for understanding downstream NMR experiments, and 4) the characterization of structural features and conformational dynamics, as well as the generation of conformational ensembles using experimental NMR data and accelerated molecular dynamics (aMD) simulations, of Alkbh5 at the major steps (apo, Zn2+ / α-KG-bound, and DNA-complexed) in the catalytic cycle necessary to decouple the structure / conformational dynamics / function relationship.

DOI

https://doi.org/10.31274/etd-20210609-152

Copyright Owner

Jeffrey Purslow

Language

en

File Format

application/pdf

File Size

154 pages

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