Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.780531
Title: Novel protein modification strategies
Author: Bower, Benjamin
ISNI:       0000 0004 7966 1724
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2018
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Abstract:
Site-directed mutagenesis allows the ready variation of proteinogenic amino acids; genetic code expansion permit the incorporation of a few more. The widespread use of these genetic techniques has caused a revolution in molecular biology. A crucial limitation of genetic techniques is the 'ribosomal filter', where an amino acid has to be processed by the ribosome or other translational enzymes. I have developed a chemical technique for the direct modification of proteins allowing the incorporation of a large scope of amino acid side-chains. Notably, this technique is independent of the ribosome. Radical addition to protein backbone dehydroalanine (Dha) forms C(sp3)-C(sp3) bonds directly onto a protein to facilitate "chemical mutagenesis". Protein glycosylation is difficult to study due to mixtures associated with biological glycosylation machinery. In Chapter 1: I have employed the above chemical mutagenesis method to create homogenous glycoproteins which retain a native bond. During this study, I have demonstrated the biological relevance of these transformations by showing reactivity with glycosidases and glycosyl transferases, found interesting reactivity of an extremely polarised amide substrate, and have used the method on an aggregation prone periplasmic protein. In Chapter 2: I used techniques developed in Chapter 1 to create intermediate mimics of the Sirt2 catalysed deacetylation of Histones. I used the mimics of Sirt2 intermediate 2 to determine the geometry around an unstable reaction intermediate. Having atomic control over the amino acid allowed us to create mimics of both the R and S diastereomers. Using analyitical size exclusion chromatography I have obtained qualitative results which show the R isomer is bound by Sirt2 much stronger than the S, hinting that the R isomer is that which is present in biology. In Chapter 3: I demonstrated a green synthesis of 2,5-dibromohexadiamide (DBHDA); which has been used extensively in the prior two chapters to modify protein Cys residues into Dha. The developed method of producing DBHDA replaces CCl4 with cyclohexane and gives a yield of 31 % over 3 steps and 6 functional group interconversions. The DBHDA produced was found to be of extremely high purity (99.7 % ± 1.21), which was determined by the use of quantitative 13C NMR spectroscopy.
Supervisor: Davis, Ben Sponsor: Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.780531  DOI: Not available
Keywords: Biochemistry ; Chemistry
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