Abstract:
Influenza A causes seasonal epidemics, and the possibility of another worldwide
influenza A pandemic remains a public health concern. The influenza A M2 protein is
implicated in the virus’s ability to bud and create new infectious virus particles.
Specifically, the C-terminal region of the virus is required for viral budding, and this
protein domain binds another influenza protein, M1. M2-M1 binding is critical to
packaging the viral genome into new virions. To probe the region of M2 involved in viral
budding and genome packing, this thesis studied the conformation of residues 60-70 of
the C-terminus via site-directed spin label electron paramagnetic resonance (SDSL-EPR)
spectroscopy. SDSL-EPR spectroscopy provided structural information about M2
reconstituted into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine : 1-palmitoyl-2-
oleoyl-sn-glycero-3-phospho-1-rac-glycerol (POPC:POPG) 4:1 lipid bilayers, a
physiologically relevant environment. Mobility and membrane accessibility data revealed
that residues 60-64 are associated with the membrane and are less mobile than residues
65-70. Residues 65-70 form a region with some periodic secondary structure, extending
into the aqueous phase. A unique contribution of this thesis was the determination of the
directionality of M2 insertion into the membrane via trypsin fragmentation and matrixassisted
laser desorption ionization-mass spectrometry (MALDI-MS). According to
current reconstitution protocols, M2 inserts into the membrane with no directionality, and
both the N- and C-termini lie at the liposome exterior.
