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Structural and biochemical characterisation of pleckstrin homology domains

Salim, Kamran; (1998) Structural and biochemical characterisation of pleckstrin homology domains. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Structural and biochemical characterisation of Pleckstrin Homology domains Pleckstrin Homology (PH) domains are modules of approximately 100-120 amino-acids found in a variety of proteins involved in intracellular signalling cascades. Although more than 90 putative PH domains have been identified by sequence analysis, a universal function for PH domains has remained elusive. However, most PH domain containing proteins appear to be localised in membrane structures. Biochemical studies have shown that some PH domains may mediate this membrane association by their ability to bind phosphoinositides. This thesis describes how data generated from structural analysis of the dynamin PH domain provided a rational basis for the identification of distinct phosphoinositide ligands for the PH domains of dynamin and Bruton's tyrosine kinase (Btk). PH domain-phosphoinositide interactions, probed using a biosensor-based assay, showed that the dynamin PH domain specifically interacted with unilamellar liposomes containing phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and, more weakly, with PtdIns 4-monophosphate. This interaction was shown to correlate with the specificity of activation of dynamin GTPase by phosphoinositides. Moreover, a dynamin mutant lacking the PH domain could not be activated by PtdIns(4,5)P2. In contrast, the PH domain of Btk specifically bound PtdIns 3,4,5-trisphosphate. Aided by molecular modelling, amino acids in the predicted phosphoinositide binding pocket of dynamin and Btk PH domains were selectively mutated in order, to identify key ligand recognition residues.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Structural and biochemical characterisation of pleckstrin homology domains
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
URI: https://discovery.ucl.ac.uk/id/eprint/10099815
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