Clostridial botulinum neurotoxins (BoNTs) induce neuroparalysis by arresting synaptic exocytosis. BoNT consists of three functional domains: protease, translocation and receptor binding, and is cleaved into two disulfide linked, polypeptide chains: Light chain (LC) protease and Heavy chain (HC) translocon and receptor binding. One of the most elusive and intriguing steps of the BoNT intoxication process is the translocation of the proteolytic LC through the BoNT protein-conducting channel of the HC, from the endosomal compartment to the cytosol. In this dissertation, we utilize single-particle electron microscopy to resolve the discrepancies in multi-domain arrangement between different isoforms of BoNT in an effort to visualize rearrangement of domains and conformational changes associated with the translocation process. We investigate the dynamics of protein- translocation focusing on the interactions between the HC channel/chaperone and its LC cargo. Single molecule translocation was monitored in real time using excised patches of neuronal cells. LC translocation requires translocation domain (TD) insertion in a receptor binding domain (RBD) dependent manner, coupled with LC unfolding and protein conduction through the TD channel. Translocation occurs as a series of progressive steps in an N- to C-terminus orientation; productive completion requires reduction of the disulfide bridge and proteolytic cleavage of the LC from the HC concurrent with LC refolding in the cytosol. The progressive, tight interplay between the individual domains inherent in the mechanism of BoNT intoxication resolve it as an elegant modular nanomachine

Repurposing drugs as treatments for COVID-19, the disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has drawn much attention. Beginning with sigma receptor ligands and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs we tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs and does not reflect specific target-based activities-rather, it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.