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Title: Biophysical and biochemical investigations of the M2 peptide of influenza A
Author: Hansen, R. K.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2003
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Abstract:
Membrane proteins, especially passive ion channels, are very difficult to characterise using normal biophysical methods. This thesis used the transmembrane domain of the M2 proton channel of Influenza A (M2-TM) as a model system for biophysical investigations. Patch clamping is the most accurate method for assessing ion channel function, but requires rigid liposomes that can withstand mechanical stress, but are not usually optimal for structural investigations. Here a new hydrogen deuterium exchange (HDX) method has been optimised for the analysis of a membrane spanning peptides in lipid vesicles. A simple electrospray ionisation mass spectrometry method is introduced for HDX studies of carefully prepared aqueous proteoliposome samples. 11 backbone amide sites of M2-TM in lipid vesicles were shown to be resistant to HDX for several weeks. By contrast, HDX of M2-TM in methanol or in detergent micelles was complete within an hour, indicating that the peptide adopts a non-nativee conformation. A solution state nuclear magnetic resonance (NMR) strategy relying on quenching the exchange reaction at low pH, lyophilisation and resuspension in acidified methanol was applied to studies of HDX in a transmembrane peptide for the first and revealed site specific information. His16 and Leu17 exchanged much faster than neighbouring residues consistent with a "flip-open" mechanism for proton conduction. Binding of the drug amantadine weakly perturbs the HDX of Ala8 and Ala9 in agreement with neutron scattering data showing an interaction with the N-terminus of the channel. NMR studies investigated details of the binding and release of fluoroamantadine to liposomes in the presence and absence of M2-TM consistent with the drug binding only to the closed state of the channel in DMPC vesicles. HDX studies have great potential to reveal details of the structure, dynamics and intermolecular interactions of membrane proteins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.603662  DOI: Not available
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