Characterization of the Membrane-Bound Topology of the Colicin E1 Channel Domain

dc.contributor.advisorMerrill, Rod
dc.contributor.authorHo, Derek
dc.date.accessioned2014-08-20T19:12:59Z
dc.date.available2014-08-20T19:12:59Z
dc.date.copyright2014-07
dc.date.created2014-07-24
dc.date.issued2014
dc.degree.departmentDepartment of Molecular and Cellular Biologyen_US
dc.degree.grantorUniversity of Guelphen_US
dc.degree.nameDoctor of Philosophyen_US
dc.degree.programmeMolecular and Cellular Biologyen_US
dc.description.abstractColicins are antimicrobial proteins produced by Escherichia coli that target susceptible bacteria in response to stressful conditions including nutrient depletion, DNA damage, over-crowding, and anaerobiosis. The C-terminal channel-forming domain of colicin E1 forms a lethal ion channel which depolarizes the cytoplasmic membrane of target bacterial cells. Prior to channel formation, the channel peptide first binds to the lipid bilayer, followed by protein unfolding and helix elongation. Finally, the channel domain adopts an insertion-competent state in which it inserts into the membrane to form the pre-channel state. The channel then opens in response to a trans-negative membrane potential and facilitates the escape of various ions from the host cells, such as Na+, K+, and H+, leading to host cell death. The objective of my thesis work was to characterize the membrane topology of the colicin E1 channel peptide using state-of-the-art fluoresence methods. Previously, researchers in our laboratory used cysteine-scanning mutagenesis to determine the surface topology and secondary structure of Helices 1 - 5 within the colicin E1 channel peptide in the pre-channel state. In this thesis work, I demonstrated that helices 6, 7 and 10 are three, distinct amphipathic α-helices which adopt a tilted topology on the membrane surface that correlates well with various methods such as harmonic analysis, fluorescence anisotropy, fluorescence emission maximum, and bilayer penetration depth. In addition, a total of twelve distances (Å) between three residues on Helix 1 (D347, S354, E361) and three Trp residues (W424, W460, W495) were estimated using fluorescence resonance energy transfer (FRET) to probe the 3-D orientation of Helix 1 relative to the rest of the channel domain in the membrane-embedded state. Furthermore, I employed the method of Schultz and co-workers to genetically encode a fluorescent amino acid (coumarin-derivative) as a FRET donor to DABMI (4-(dimethylamino)phenylazophenyl-4-maleimide) as the acceptor. This work provided six more interhelical distances between Helices 1 – 6 of the channel peptide in the pre-channel state. Overall, this work has provided a number of constraints towards the development of an improved colicin E1 model (umbrella model) that accounts for all of the FRET data for the pre-channel state.en_US
dc.description.sponsorshipNatural Sciences and Engineering Research Council of Canada
dc.identifier.urihttp://hdl.handle.net/10214/8307
dc.language.isoenen_US
dc.publisherUniversity of Guelphen_US
dc.rightsAttribution-NonCommercial-NoDerivs 2.5 Canada*
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/2.5/ca/*
dc.subjectColicin E1en_US
dc.titleCharacterization of the Membrane-Bound Topology of the Colicin E1 Channel Domainen_US
dc.typeThesisen_US

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