Spearpoint, PA;
(2008)
Structure-function studies of the influenza A M2 proton channel.
Doctoral thesis , UCL (University College London).
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Abstract
The influenza A virus M2 protein is a minor component of the virus membrane, it forms a homotetrameric, pH-activated, proton-selective channel and is the target of the anti-influenza drugs, amantadine and rimantadine. Two projects were undertaken to study the structure-function relationships of M2 and the mechanism of its inhibition. Firstly, an M2 protein with a pH-sensitive GFP fused to the C-terminus was constructed to determine whether it could be used to measure directly M2 proton conduction. The pH probe was responsive to rimantadine-sensitive changes in the pH gradient which were detected but were small. This was in part due to reduced proton conductance of the protein possibly due to altered conformation of the M2 pore, but may also reflect the low sensitivity of the GFP probe to local pH in the vicinity of the plasma membrane. Secondly tryptophan fluorescence studies of purified, E.coli- expressed M2 protein in detergent micelles investigated the mechanism of proton conduction and inhibition by anti-M2 drugs. Shifts in emission maxima and fluorescence intensity measurements relating to the tryptophans in the M2 pore indicated a molecular interaction between histidine 37 (His37) and tryptophan 41 (Trp41). pKa values from these experiments correlated with previously reported proton-mediated activation and permeation of the M2 channel. Acrylamide quenching showed that upon histidine protonation, Trp41 was more solvent accessible while polarisation measurements indicated a more restricted environment for Trp41 at low pH. Red-edge excitation measurements suggested a proteinaceous pore in which water molecules were highly immobile when the channel was closed, yet could freely reorientate upon channel activation. The M2 inhibitor rimantadine reversed these phenomena either fully or partially. Equilibrium and time-resolved measurements of this drug-induced reversal permitted the determination of both association and dissociation rate constants and the affinities of drug interaction with M2. These results provide further support for an allosteric mechanism of amantadine inhibition.
| Type: | Thesis (Doctoral) |
|---|---|
| Title: | Structure-function studies of the influenza A M2 proton channel |
| Identifier: | PQ ETD:593448 |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Thesis digitised by ProQuest. Third party copyright material has been removed from the ethesis. Images identifying individuals have been redacted or partially redacted to protect their identity. |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1446119 |
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