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Characterising side-chain motions in proteins by Nuclear Magnetic Resonance and Molecular Dynamics to report on function and regulation

Siemons, Lucas; (2020) Characterising side-chain motions in proteins by Nuclear Magnetic Resonance and Molecular Dynamics to report on function and regulation. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Analysing the motions proteins undergo is vital for understanding a wide variety of biological processes. In particular side chains provide a wide range of chemical groups allowing proteins to carry out diverse functions such as catalysis and regulating gene expression. A key theme in this thesis is understanding the roles side-chains play in protein dynamics. To do this we use molecular dynamics, density functional theory and nuclear magnetic resonance. The first part of this work describes the relationship between the isoleucine side- chain conformation and chemical shift. We show there is a clear dependence between the χ angles and the observed side-chain’s 13C chemical shifts. This relationship is then used to determine rotamer distributions in the L24A FF domain’s excited state and the 42 kDa membrane complex DsbA-DsbB. In addition we use our methodology to show that the isoleucine random coil distribution in two model peptides is substantially different to the statistical distribution derived from the PDB. The second part of this thesis focuses on characterising the dynamic processes reg- ulating histone deacetylase 8. Here two approaches are used. The first concentrates on molecular dynamics to show the allosteric connection between the active site, the bind- ing rail and I19, a naturally occurring mutation site in patients. In conjunction with this we aimed to carry out a backbone independent methyl assignment. To aid joining intra- residue methyls we developed the HMBC-HMQC that utilises scalar coupling based transfers. This has many advantages over NOE based approaches as it directly reports on the bonding network, greatly simplifying the interpretation of crowded regions of the spectra. In addition to this we also made substantial progress towards assigning the ILV methyls by determining the residue types, joining intra-residue methyls and building an NOE network between the observed resonances.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Characterising side-chain motions in proteins by Nuclear Magnetic Resonance and Molecular Dynamics to report on function and regulation
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2020. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/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.
Keywords: Nuclear Magnetics Resonance, NMR, HDAC8, Molecular Dynamics, MD, chemical shift, isoleucine, rotamers
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
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 > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
URI: https://discovery.ucl.ac.uk/id/eprint/10094856
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