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Title: Post-translational mutagenesis : radical methods for protein modification
Author: Wright, Tom
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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The naturally occurring post-translational modification of proteins expands the chemical and structural diversity available for protein function. Enzymatic processes are known to modify proteins after translation, and in many cases these modifications have been demonstrated to be critical for biological function. However, the paucity of methods for introduction of site-specific modifications to proteins remains a key obstacle to their biochemical study. Access to these naturally modified proteins has long been complicated by their low abundance, difficulties in purifying homogeneous samples from cellular samples and the intractability of bacterial expression systems towards post-translational modification. In this thesis, we report an important step towards circumventing these issues by using a novel 'tag and modify' chemical strategy, termed 'post-translational mutagenesis'. We describe the development and application of C(sp3)-C(sp3) bond-forming reactions on proteins under biocompatible conditions, exploiting unusual free-radical chemistry to form a wide diversity of protein side-chains. Natural, unnatural, posttranslationally-modified (methylated, glycosylated, phosphorylated, hydroxylated) and labeled (fluorinated, isotopically-labeled) side-chains have been installed on a number of proteins. Extensive characterisation of modification site, connectivity and modified protein structure and function confirm the benign nature of our protocols. The modified proteins thus produced have been used to investigate novel aspects of epigenetic regulation of histone proteins, in the context of free histones and in nucleosome particles, and as novel 'isotope PTMs' for protein characterisation by NMR. We believe this approach, a form of 'protein editing', will have wide application in biochemistry and synthetic biology for accessing diverse modified proteins for research.
Supervisor: Davis, Ben Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available