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Title: Photochemical harpoons : covalent labels for multi-protein complexes
Author: Smida, F. A.
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2013
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The identification of the biomolecular interaction partners of small bioactive molecules is a fundamental problem in drug discovery and cell biology. This thesis describes the development of fluorescent chemical probes to identify the biomolecular targets of the known organophosphate toxin, phenyl saligenin phosphate (PSP), and the cardioprotective agent diazoxide. PSP is an organophosphate toxin that irreversibly inhibits hydrolase enzymes such as trypsin and chymotrypsin along with the common organophosphate target acetylcholine esterase. PSP is also suspected of affecting many other cell functions and may interact with a large number of cellular proteins. In this work phenyl saligenin phosphate has been synthesised and its inhibitory effect on the action of transglutaminase 2 (TGase2) demonstrated. Analogues of PSP containing an attached dansyl amide fluorescent group have been prepared and incubated with purified enzymes trypsin, chymotrypsin and TGase2. SDS-PAGE analysis demonstrates effective fluorescent labelling and a covalent interaction between the toxin analogue and the enzymes. The KATP channel opener, diazoxide displays marked cardioprotective effects and is reported to bind to mitochondrial KATP channels. However, the molecular structure of these channels is still largely unknown. This thesis describes the design and the synthesis of a chemical tool to covalently attach fluorescently labels to the proteins which will bind diazoxide. Chemical tools for fluorescent labelling of diazoxide targeted proteins have been prepared. Each consists of a photochemically activated reactive ‘barb’ and coupled fluorescent component linked to modified diazoxide bait. In developing these molecules, a range of functionalised diazoxide bait components were prepared and tested for retained biological activity compared to the parent compound. Two active analogues were linked to either benzophenone or diazirine (photoreactive) and dansyl amide (fluorescent) components. The non-specific photochemical reactivity of these labelling compounds with bovine serine albumin was established. The incubation and photolysis with mitochondrial extracts showed selective photo labelling of only three biomolecular components. The identification of these biomolecules is the subject of on-going investigation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available