Mechanism of Tetrameric Transmembrane Influenza A/M2 Proton Channel Activation Open Access

Zawadzki, Jonathan (2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/x633f180f?locale=en
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Abstract

Influenza is a common infection caused by the influenza virus, presentation of which can vary from the fever and headache of the contemporary Avian flu to the fatal Spanish flu pandemic of 1918. Replication of this virus is dependent on increased proton conductance through an activated viral transmembrane M2 proton channel, a 96-residue homotetramer. In order to develop methods to control influenza replication, the proton channel's structure and function have been intensively studied. The primary objective of this study was to observe clear evidence for proton channel conformational change in which the C-termini dilates when exposed to low biologic pH (pH 4.5-6). This was accomplished using Forster Resonance Energy Transfer (FRET) analysis on donor and acceptor fluorophore labeled C-termini to determine dilation change. With a pH drop from 6.0 to 4.5, the C-termini were found to dilate from 3.0 to 3.4 nm in length, presumably allowing increased proton transport. Understanding the mechanism by which the structure of M2 proton channel activates in lower pH ranges may help develop new treatments for influenza infections.

Table of Contents

I. Introduction .....................................................................................................................1

M2 Proton Channel and Influenza A Virus…….……….....................................................1

Structure and Function of M2 Transmembrane Domain…………………….……………2

FRET: Forster Resonance Energy Transfer.........................................................................4

M2 Proton Channel Conformation Change Measured by FRET……….….....…...………5

Purpose ................................................................................................................................7

II. Experimental ....................................................................................................................7

Peptide Synthesis and Cleavage .........................................................................................7

Optimal FRET Pair ……….................................................................................................8

Labeling Peptide with Dye Molecules.................................................................................9

Mass Spec on Finished Labeled Peptide...........................................................................10

M2 Recombination to Membrane ……………………………………………………….11

Fluorescence Measurement...............................................................................................12

III. Results and Discussion ..................................................................................................12

Absorbance of Free Dye in Different pH Environments..................................................12

Tetramerization and pH Dependence of M2 Proton Channel..........................................13

Control experiment: N-termini labeled M2………………………...……………......….16

FRET Efficiency and Distance.........................................................................................17

IV. Discussion and Improvements to the Experimental Procedures……………..……..24

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