Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711723
Title: Synthetic phosphorylation of kinases for functional studies in vitro
Author: Chooi, Kok Phin
ISNI:       0000 0004 6060 4236
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2014
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Abstract:
The activity of protein kinases is heavily dependent on the phosphorylation state of the protein. Kinase phosphorylation states have been prepared through biological or enzymatic means for biochemical evaluation, but the use of protein chemical modification as an investigative tool has not been addressed. By chemically reacting a genetically encoded cysteine, phosphocysteine was installed via dehydroalanine as a reactive intermediate. The installed phosphocysteine was intended as a surrogate to the naturally occurring phosphothreonine or phosphoserine of a phosphorylated protein kinase. Two model protein kinases were investigated on: MEK1 and p38α. The development of suitable protein variants and suitable reaction conditions on these two proteins is discussed in turn and in detail, resulting in p38α-pCys180 and MEK1-pCys222. Designed to be mimics of the naturally occurring p38α-pThr180 and MEK1-pSer222, these two chemically modified proteins were studied for their biological function. The core biological studies entailed the determination of enzymatic activity of both modified proteins, and included the necessary controls against their active counterparts. In addition, the studies on p38α-pCys180 also included a more detailed quantification of enzymatic activity, and the behaviour of this modified protein against known inhibitors of p38α was also investigated. Both modified proteins were shown to be enzymatically active and behave similarly to corresponding active species. The adaptation of mass spectrometry methods to handle the majority of project's analytical requirements, from monitoring chemical transformations to following enzyme kinetics was instrumental in making these studies feasible. The details of these technical developments are interwoven into the scientific discussion. Also included in this thesis is an introduction to the mechanism and function of protein kinases, and on the protein chemistry methods employed. The work is concluded with a projection of implications that this protein chemical modification technique has on kinase biomedical research.
Supervisor: Jones, Lyn H. ; Davis, Benjamin G. Sponsor: Engineering and Physical Sciences Research Council ; Pfizer Ltd
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.711723  DOI: Not available
Keywords: Biomimetics ; Biochemistry ; Organic chemistry ; Chemical biology ; Protein chemistry ; Biomolecular engineering ; Mass spectrometry ; MEK1 ; Cysteine ; Enzyme inhibition ; Protein chemical modification ; Phosphorylation ; Enzyme kinetics ; Protein mass spectrometry ; Site-selective protein modification ; Protein kinases ; Phosphocysteine ; p38α ; Biomolecular recognition ; Enzymatic activity
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