Imaging the assembly of the Staphylococcal pore-forming toxin alpha-Hemolysin

Thesis

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.

Authors: Thompson, J; James R. Thompson

• Publication date: 2009
• DOI: 10.5287/ora-vjqnmvjvx
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: Oxford University, UK
• Language: English
• Keywords: pore-forming toxin; droplet bilayers; electrophysiology; single-molecule fluorescence; Staphylococcus aureus; alpha-Hemolysin; lipid bilayers; single-channel electrical recording; droplet-on-hydrogel bilayers; assembly
• Subjects: Physical & theoretical chemistry; Chemical biology; Biochemistry; Condensed Matter Physics; Biosensors; Laser Spectroscopy; Molecular biophysics (biochemistry); Protein chemistry; Protein folding; Biophysical chemistry; Infectious diseases; Membrane proteins; Haematology; Nano-biotechnology; Biophysics; High resolution microscopy