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Using NMR Spectroscopy to study the structural dynamics and inhibition of aggregation of TAR DNA binding protein-43

Alam, Sarah Masood; (2022) Using NMR Spectroscopy to study the structural dynamics and inhibition of aggregation of TAR DNA binding protein-43. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

This thesis focuses on understanding the role of trans-activation response DNA binding protein-43 (TDP-43) which has been identified as the major pathological protein of Amyotrophic Lateral Sclerosis (ALS), but it is currently not known exactly how this protein is involved in the pathogenesis of the disease. TDP-43 is a 414-residue, multi-domain protein and its disordered C-terminal (CTD274-414) is thought to propagate its aggregation, although a mechanistic understanding, and indeed, structural information on TDP-43 overall are lacking. NMR spectroscopy is unique in its capacity to study dynamic systems at near-atomic resolution and is well-suited to study intrinsically disordered domains. Using in vitro and in-cell NMR spectroscopy, this thesis aims to understand the processes by which it transitions from a functionally active protein to toxic intracellular aggregates. This work describes the development of an expression and purification strategy of TDP-43 under native conditions. NMR studies were carried out in isolation and inside living cells using in-cell NMR in human cells which is an invaluable tool for residue-specific, structural and function investigation of isotopically labeled proteins inside a biologically relevant environment. Riluzole is the first FDA-approved medication for ALS although its mechanism of action is not fully understood. High resolution NMR studies were performed to provide novel insights into the interaction between CTD274-414 and riluzole. Biochemical and biophysical assays were developed and performed to investigate the effect on the aggregation properties of CTD274-414 by riluzole. These experiments are forming the basis for studying how the other domains of TDP-43 are involved in its aggregation and to identify other potential small molecule aggregation inhibitors.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Using NMR Spectroscopy to study the structural dynamics and inhibition of aggregation of TAR DNA binding protein-43
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2022. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL
URI: https://discovery.ucl.ac.uk/id/eprint/10143959
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