We use cookies to ensure that we give you the best experience on our website. By continuing to browse this repository, you give consent for essential cookies to be used. You can read more about our Privacy and Cookie Policy.

Durham e-Theses
You are in:

Investigation of a Histidine-Based Probe for the Exploration of Proteomes

BUERDSELL, MICHAELA,LOUISE (2022) Investigation of a Histidine-Based Probe for the Exploration of Proteomes. Doctoral thesis, Durham University.

PDF - Accepted Version


Leishmaniasis is a neglected tropical disease which affects 0.7-1 million people per year. Current chemotherapies for leishmaniasis are toxic with long treatment times and reports of increasing resistance, which stresses the importance of this research area. Inositol phosphorylceramide synthase is a membrane bound enzyme that has no direct human homologue, which converts ceramide to inositol phosphorylceramide through the action of a highly conserved HHD catalytic triad. An ideal method to study this enzyme further would be through activity-based protein profiling, however, there are currently no activity-based probes reported that reacts with this type of active site. Therefore, an activity-based probe was designed based on the structure of diethyl pyrocarbonate, a compound known to bind covalently to active site histidine residues. The synthesised activity-based probe was shown to inhibit Leishmania major inositol phosphorylceramide synthase in a simple assay. In addition, the probe was shown to selectively bind to the active site histidine residue in two pure enzyme models; one of which has the same catalytic triad as inositol phosphorylceramide synthase, and the other was an acid base active site histidine residue. Further, this activity-based probe was able to isolate an overexpressed enzyme in the lysate of Escherichia coli as well as bind to intrinsic proteins. Following the function validation of the activity-based probe, preliminary work was started in Leishmania to isolate proteins identify expressed enzymes.

Item Type:Thesis (Doctoral)
Award:Doctor of Philosophy
Keywords:Activity based protein profiling, Activity based probes, histidine
Faculty and Department:Faculty of Science > Chemistry, Department of
Thesis Date:2022
Copyright:Copyright of this thesis is held by the author
Deposited On:21 Apr 2022 10:38

Social bookmarking: del.icio.usConnoteaBibSonomyCiteULikeFacebookTwitter