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Mapping Functionally Important and Stabilising Regions in Biotherapeutic Proteins, using NMR and Mutagenesis

Kellerman, Mark-Adam Walter; (2023) Mapping Functionally Important and Stabilising Regions in Biotherapeutic Proteins, using NMR and Mutagenesis. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Structure-function relationships in proteins refer to a trade-off between stability and bioactivity, moulded by evolution of the molecule. Identifying which protein amino acid residues jeopardise global or local stability for the benefit of bioactivity would reveal residues pivotal to this structure-function trade-off. Demonstrated here is the use of varied-temperature 15N-1H heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy to probe the microenvironment and dynamics of residues in granulocyte-colony stimulating factor (G-CSF). This experimental approach was also used to investigate (de-) stabilising mechanisms of action for previously studied excipients with G-CSF. Combining NMR with in silico analysis revealed four structural clusters that are subject to localised conformational changes (some of which are key to bioactivity) or partial unfolding prior to global unfolding at higher temperatures. Mechanisms by which excipients influence these important structural changes and implement their own structural clusters reflects their impact on stability and function. This approach was leveraged for semi-rational mutant/formulation design. These mutants were tested for fitness with respect to thermostability and functionality. The Mutants P65V and E45Q were constructed to elicit mutation-excipient interactions, and presented the largest impact on the respective fitness. Hence, this study proposes an approach to profile residues, thus highlighting their roles in stability and bioactivity while exposing potential mutation-excipient interactions. This permits a semi-rational protein engineering approach to optimise desirable protein fitness characteristics.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Mapping Functionally Important and Stabilising Regions in Biotherapeutic Proteins, using NMR and Mutagenesis
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2023. 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
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering
URI: https://discovery.ucl.ac.uk/id/eprint/10169173
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