COMING SOON! PQDT Open is getting a new home!

ProQuest Open Access Dissertations & Theses will remain freely available as part of a new and enhanced search experience at

Questions? Please refer to this FAQ.

Dissertation/Thesis Abstract

Crystallographic and NMR Studies of Mutant Tetramer α-Synuclein and Enzymatic and NMR Studies of Human Iron Acireductone Dioxygenase
by Liu, Xinyue, Ph.D., Brandeis University, 2020, 154; 28092870
Abstract (Summary)

Parkinson’s disease (PD) is the second most common neurodegenerative disease. Currently approximately one million people in the U.S and more than 10 million people worldwide are living with PD. Lewy bodies are the pathologic hallmark of PD. The protein α-synuclein has been identified as the major component of Lewy bodies and Lewy neurites. Chapter 1 provides an introduction to α-synuclein, from the widely believed intrinsically disordered monomer narrative to increasing recent evidence on α-synuclein adopting a stable multimeric (primarily tetrameric) form in solution. We believe that the means by which heterologously expressed α-synuclein was purified likely disrupts the tetrameric form and contributed to the intrinsically disordered monomer narrative. Inspired by the tetrameric domain of the Nipah virus RNA phosphoprotein, site directed mutagenesis was used to mutate valines to leucines in selected position (V52/55L, V74/77L, V52/55/74/77L) within the hydrophobic core of the tetramerization domain, to encourage the formation of leucine zippers in the hope of achieving a crystallization stable dynamic tetrameric structure. The purified mutant proteins were characterized using native gel and size exclusion chromatography. Subsequently, analysis on how buffer conditions were to affect leucine mutants using ThermoFluor aggregation assays were performed to find possible crystallization conditions. Lastly, Chapter I describes crystallography efforts and mutant NMR backbone assignments with suggestions for future research directions on this project.

A participant in the methionine salvage pathway, acireductone dioxygenase (ARD) has two isozyme forms that perform different chemistry depending on its incorporated metal center at the active site. Such dual chemistry has been observed in both bacterial and mammalian systems. Chapter II describes the solution structural NMR characterization of the Fe2+-bound human acireductone dioxygenase (HsARD) along with the interaction of HsARD with the regulatory tail of membrane type-1 matrix metalloproteinase (MT1-MMP) as measured by fluorescence polarization (FP) titration assay, isothermal calorimetry (ITC) and NMR. The Fe2+-bound HsARD NMR structure were compared to the Ni2+ -bound HsARD NMR structure to highlight the consistency in structure to perform dual chemistry observed in other bacterial and mammalian systems. Discussions on how the last 20 residues on MT1-MMP were to interact with both Fe2+- bound HsARD and Ni2+ -bound HsARD raises questions for further investigation.

Indexing (document details)
Advisor: Pochapsky, Thomas C.
Commitee: Pandelia, Maria-Eirini, Ringe, Dagmar
School: Brandeis University
Department: Chemistry
School Location: United States -- Massachusetts
Source: DAI-B 82/3(E), Dissertation Abstracts International
Subjects: Biochemistry, Chemistry, Biophysics
Keywords: Acireductone dioxygenase, Crystallography, NMR, Parkinson’s disease, Type-1 matrix metalloproteinase, α-synuclein
Publication Number: 28092870
ISBN: 9798664798579
Copyright © 2021 ProQuest LLC. All rights reserved. Terms and Conditions Privacy Policy Cookie Policy