Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.504611
Title: Imaging the assembly of the Staphylococcal pore-forming toxin alpha-Hemolysin
Author: Thompson, James Russell
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2009
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Abstract:
Alpha-hemolysin is a pore-forming toxin secreted by pathogenic Staphylococcus aureus. Its spontaneous oligomerization and assembly into a trans-bilayer beta-barrel pore is a model for the assembly of many other pore-forming toxins. It is studied here in vitro as a means to probe general membrane protein oligomerization and lipid bilayer insertion. This thesis details the results of experiments to develop and implement a novel in vitro lipid bilayer system, Droplet-on-Hydrogel Bilayers (DHBs) for the single-molecule imaging of alpha-hemolysin assembly. Chapter 2 describes the development of DHBs and their electrical characterization. Experiments show the detection of membrane channels in SDS-PAGE gels post-electrophoresis and DHBs use as a platform for nanopore stochastic sensing. Chapter 3 describes the engineering and characterization of fluorescently-labelled monomeric alpha-hemolysin for use in protein assembly imaging experiments described in Chapter 6. Chapter 4 describes the characterization of DHB lipid fluidity and suitability for single-molecule studies of membrane protein diffusion. In addition, a novel single-particle tracking algorithm is described. Chapter 5 describes experiments demonstrating simultaneous electrical and fluorescence measurements of alpha-hemolysin pores embedded within DHBs. The first multiple-pore stochastic sensing in a single-lipid bilayer is also described. Chapter 6 describes experiments studying the assembly of alpha-hemolysin monomers in DHBs. Results show that alpha-hemolysin assembles rapidly into its oligomeric state, with no detection of long-lived intermediate states.
Supervisor: Wallace, Mark Ian Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.504611  DOI: Not available
Keywords: Biochemistry ; Molecular biophysics (biochemistry) ; Nano-biotechnology ; Infectious diseases ; Haematology ; Biophysical chemistry ; Biophysics ; Biosensors ; Chemical biology ; Laser Spectroscopy ; Membrane proteins ; Physical & theoretical chemistry ; Protein chemistry ; Protein folding ; High resolution microscopy ; Condensed Matter Physics ; alpha-Hemolysin ; Staphylococcus aureus ; pore-forming toxin ; assembly ; single-molecule fluorescence ; single-channel electrical recording ; electrophysiology ; droplet bilayers ; droplet-on-hydrogel bilayers ; lipid bilayers
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