Conformational Studies of a Domain of the Influenza A M2 Protein Involved in Viral Budding and Morphology

In my two years working in the Howard lab, I have cycled through a number of projects. These projects fall under the broad context of studying of how the cytoplasmic tail of a particular membrane protein of the influenza A virus, the Matrix protein 2 (M2), is involved in viral budding. I have chosen to present each project in the form of an independent manuscript, with separate Introduction, Experimental, Results, and Discussion sections. Some broader background and theory is briefly provided in Chapters 1 and 2, but Chapters 3-5 are designed to be largely self-contained. Potential future directions pertaining to all three projects are collected in Chapter 6. Science is a collaborative enterprise, and this thesis has benefited from intellectual and experimental contributions from a number of talented collaborators. For the chapters where data is presented (Chapters 3-5), an explanation of the contributions of others is described in a statement at the end of the chapter

The Distal Cytoplasmic Tail Of The Influenza A M2 Protein Dynamically Extends From The Membrane

The influenza A M2 protein is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle. The monomeric subunits of the M2 homotetramer consist of an N-terminal ectodomain, a transmembrane domain, and a C-terminal cytoplasmic domain. The transmembrane domain forms a four-helix proton channel that promotes uncoating of virions upon host cell entry. The membrane-proximal region of the C-terminal domain forms a surface-associated amphipathic helix necessary for viral budding. The structure of the remaining ~34 residues of the distal cytoplasmic tail has yet to be fully characterized despite the functional significance of this region for influenza infectivity. Here, we extend structural and dynamic studies of the poorly characterized M2 cytoplasmic tail. We used SDSL-EPR to collect site-specific information on the mobility, solvent accessibility, and conformational properties of residues 61–70 of the full-length, cell-expressed M2 protein reconstituted into liposomes. Our analysis is consistent with the predominant population of the C-terminal tail dynamically extending away from the membranes surface into the aqueous medium. These findings provide insight into the hypothesis that the C-terminal domain serves as a sensor that regulates how M2 protein participates in critical events in the viral infection cycle

Site-Directed Spin Labeling EPR Spectroscopy Of The Cytoplasmic Tail Of Influenza A M2

The M2 protein is a 97 residue homotetrameric, multifunctional ion channel that plays critical roles during the influenza infection cycle. While a variety of high-resolution biophysical techniques have been used to characterize the transmembrane domain (residues 22-46) and the juxtamembrane C-terminal region (46-62), less is known about the conformation and dynamics of the remaining residues of the C-terminal cytoplasmic tail. Here, we use site-directed spin labeling electron paramagnetic spectroscopy (SDSL-EPR) experiments to probe the secondary structure and membrane topology of cytoplasmic tail residues 60-80 when the protein is reconstituted into lipid bilayers. Cholesterol is essential for the role the C-terminal domain of the M2 protein plays in viral budding. SDSL-EPR data is collected in both in the presence and absence of cholesterol