eprintid: 10094139 rev_number: 29 eprint_status: archive userid: 608 dir: disk0/10/09/41/39 datestamp: 2020-05-21 08:40:03 lastmod: 2021-04-01 06:11:03 status_changed: 2020-05-21 08:40:03 type: thesis metadata_visibility: show creators_name: Wilkinson, Henry C. title: Exploration and exploitation of non-canonical amino acid incorporation to detect or improve transketolase activity and stability ispublished: unpub divisions: UCL divisions: A01 divisions: B04 divisions: C05 divisions: F43 keywords: non-canonical amino acid incorporation, transketolase, Enzyme stability, Enzyme activity, Cofactor binding note: 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. abstract: Site-specific, non-canonical amino acid (ncAA) incorporation into proteins is a ground-breaking methodology that facilitates the exploration of protein sequence space beyond what is possible naturally, and enables the site-specific introduction of bio-orthogonal functional groups. NcAAs can be exploited to improve, modify or create novel enzyme activities, or to probe protein structure- function relationships through genetically encoded or post-translationally modified ncAAs. A transketolase-based ncAA-incorporation platform based on an existing ncAA-incorporation plasmid, pUltra, was developed and optimised to form the foundation of this study. The thesis outlines novel approaches to quantitatively analyse incorporation fidelity. and to adjust activity parameters to account for misincorporation. The platform was first utilised to incorporate para-substituted phenylalanine derivatives into the active site of a transketolase variant, S385Y/D469T/R520Q, previously evolved to accept aromatic substrates, to probe the effect of side-chain structure and electronics on catalytic activity, acceptor substrate inhibition, enzyme stability and binding pocket preference. A novel function, the Modified Michaelis-Menten function, was derived to describe and quantify the extensive substrate inhibition observed at high [substrate]. To this author’s knowledge, variant pAMF is the first example in which catalytic activity (2.4-fold and 2.6-fold improvements in Km and catalytic efficiency) and stability (5 oC increase in Tm) have been simultaneously evolved via site-specific incorporation of ncAAs into an active site. After briefly exploring the use of para-cyanophenylalanine (pCNF) as a genetically-encoded fluorescent probe, it became apparent that transketolase intrinsic fluorescence could be used to determine the binding parameters of the transketolase cofactors and substrates, independently of activity, in a novel TPP-binding assay. A second, previously uncharacterised, low-affinity TPP binding-site was observed that belonged to an inactive, low-affinity TK species, TKlow. The two forms of the monomeric unit with high- (TKhigh) and low- (TKlow) affinity could dimerise to form three compositions of dimer. This work led to the proposal of a novel Two-Species Model of transketolase activation, regulation and inhibition that describes the interconversions between apo-/holo- TKhigh and TKlow in response to heat-shock and oxidative stress, and its physiological relevance. This is a significant discovery in a well-studied protein and could have major implications in both transketolase research and in a wider context, such as in cancer research. 4 ABSTRACT Finally, a novel, FRET-based stability assay was developed that detected local protein unfolding and aggregation of transketolase, when both purified and in cell lysate, via incorporation and bio-orthogonal labelling of the non-canonical amino acid para-azidophenylalanine (pAzF). The stability assay was applied to transketolase in a lysate background to investigate the effect of cell- based macromolecular crowding on local stability. While the ncAA pAzF and the two Alexafluor dyes have been used previously to interrogate protein structure and stability under different conditions, to this author’s knowledge this is the first time doubly-incorporated and labelled pAzF has been used to study unfolding in real time via FRET, both in purified protein and in a lysate background. date: 2020-03-28 date_type: published oa_status: green full_text_type: other thesis_class: doctoral_open thesis_award: Ph.D language: eng thesis_view: UCL_Thesis primo: open primo_central: open_green verified: verified_manual elements_id: 1773943 lyricists_name: Wilkinson, Henry lyricists_id: HCWIL75 actors_name: Wilkinson, Henry actors_name: Allington-Smith, Dominic actors_id: HCWIL75 actors_id: DAALL44 actors_role: owner actors_role: impersonator full_text_status: public pages: 190 event_title: UCL (University College London) institution: UCL (University College London) department: Biochemical Engineering thesis_type: Doctoral editors_name: Dalby, P citation: Wilkinson, Henry C.; (2020) Exploration and exploitation of non-canonical amino acid incorporation to detect or improve transketolase activity and stability. Doctoral thesis (Ph.D), UCL (University College London). Green open access document_url: https://discovery-pp.ucl.ac.uk/id/eprint/10094139/13/Wilkinson_10094139_thesis_id_removed.pdf