Influenza A virus is a major public health concern because of its annual death toll and its potential to cause devastating pandemics. Fundamental information on the life cycle of the influenza virus is still being discovered which could lead to new tactics for treating influenza infections. Recently a series of studies on the role of the influenza A M2 protein in virus assembly and budding have been published. This proposal describes experiments designed to understand the conformation and dynamics of the C-terminal juxtamembrane region of the M2 protein shown to be critical to viral budding. The majority of previously published biophysical work on M2 has focused on truncated M2 peptide constructs solubilized in detergent or in bilayers of limited physiological relevance. The full-length protein can now be efficiently over-expressed and purified. To eliminate potential artifacts due to the use of fragments of M2 and distortion of the structure due to non-native hydrophobic environments, the work proposed here will use full-length protein reconstituted into membrane bilayers that reflect the lipid composition of influenza A virions as well as model membrane bilayers in which viral budding functional assays have been carried out. An atomic level understanding of the role M2 plays in viral budding requires detailed structural information. Our strategy for characterizing the structure and dynamics of the C-terminal juxtamembrane region of the full-length influenza A M2 protein will rely on site-directed spin label (SDSL) EPR experiments. We will prepare a series of full-length M2 proteins with spin labels in the C- region (residues 46-62) shown previously to play an essential role in viral budding. SDSL EPR data used in structure determination will include patterns of spin label mobilities, distance constraint from spin-spin couplings and accessibility to paramagnetic reagents of varying bilayer/aqueous solubility. Using deuterated forms of cholesterol, electron spin-echo envelope modulation (ESEEM) experiments will be used to measure distances between the deuterium nuclei on cholesterol and spin-labeled sites on the M2 protein. The high sensitivity of SDSL EPR studies will allow a range of cholesterol:peptide:lipid ratios to be tested, enabling a fuller characterization of the location(s) and affinities of cholesterol binding than has been previously possible.