Keating, Patrick MarcellusLassa virus (LASV) is the most prevalent member of the arenavirus family and the causative agent of Lassa fever, a viral hemorrhagic fever. Although there are annual outbreaks in West Africa and recently isolated cases worldwide, no current therapeutics or vaccines pose LASV as a significant global public health threat. One of the key steps in LASV infection is the delivery of its genetic material by fusing its viral membrane with the host cell membrane. This process is facilitated by significant conformational changes within glycoprotein 2 (GP2), yielding distinct prefusion and postfusion structural states. However, structural information is missing to understand the changes that occur in the transmembrane domain (TM) during the fusion process. Investigating how the TM participates in membrane fusion will provide new insights into the LASV fusion mechanism and uncover a new therapeutic target sight to combat the dangerous infection. Here, we describe our protocols for expressing and purifying the isolated TM and our GP2 constructs which we use to probe the relationship between the structure of the TM and its influence on the function of GP2.We express TM as a fusion protein with a Hisx9 tag and a TrpLE tag using E. coli bacterial cells. We purify the TM using Nickel affinity chromatography and enzymatic cleavage to remove the tags. Since the TM is prone to aggregation, we must use a strong denaturant, trichloroacetic acid (TCA), to remove the TM from the resin. The isolated TM is then buffer exchanged to a detergent solution for structural studies. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, our structural studies revealed a pH-dependent structural change resulting in an N-terminal extension of the alpha helix in the postfusion state. To test the importance of this structural change, we used the GP2 construct to perform a modified lipid mixing fusion assay. Our results from the fusion assay and a combined mutational study revealed that this structural change is important for the fusion efficiency of GP2. Loss of this extension resulted in lower fusion activity. To further understand these structural changes and to probe the TM’s environmental interactions, we turned to fluorine NMR. This method gives us a unique and highly sensitive probe to monitor changes in the structure and membrane environment. We describe our incorporation protocol of fluorine into the TM and our method for incorporating the TM into a lipid bilayer system. We describe preliminary results showing sensitive changes in the structure of the TM and the implications this method has to enhance our understanding of the LASV membrane fusion mechanism.enDissertationBiochemistryBiophysicsGlycoproteinLassa virusMembrane fusionNMRpHTransmembrane domain