The abundant neuronal protein a-synuclein, which is implicated in neurodegenerative disease, adopts a wide variety of conformational states that contribute to its physiological and pathological activities. We recently used deep mutational scanning to probe the conformation of a-synuclein that drives its toxicity in a cellular model, and surprisingly, we found that this aberrant phenotype is driven by a dynamic, membrane-bound amphiphilic helix, which is also believed to mediate its native physiological role in vesicle trafficking. How does the physiological conformation of a-synuclein contribute to pathology? Our data highlight a critical sequence feature of a-synuclein that we hypothesize mediates both its physiological and pathological interactions with lipid membranes by increasing dynamics of the membrane-bound helix. In this proposal, we will test our hypothesis for the molecular basis of helix dynamics, as well as the contribution of those dynamics to exocytosis and aggregation. In order to test these hypotheses, the principal investigator (PI) requires additional training in nuclear magnetic resonance (NMR) spectroscopy, mammalian cell culture and manipulation, and cellular imaging, as well as additional expertise in membrane protein biophysics and the molecular biology of vesicle trafficking and neurotransmission. These hypotheses will therefore be addressed under the mentorship of Prof. William DeGrado, one of the world’s leading experts in membrane protein structure and function, and Prof. Robert Edwards, one of the world’s leading experts on the role of a-synuclein in exocytosis. With their guidance, the PI will (1) test the contribution of a-synuclein sequence features to the dynamics of the membrane bound state using NMR spectroscopy, and (2) test the role of those sequence features in mediating a-synuclein’s effect on exocytosis in neuroendocrine cells. Following completion of the mentored phase, the PI will (3) test the contributions of dynamic membrane binding to a-synuclein aggregation using deep mutational scanning. Together, these aims will provide a molecular mechanism by which the unique structure of a-synuclein contributes to both its physiological and pathological activities. Moreover, the training provided by these experiences will position the PI to launch an independent scientific career examining functional interactions between proteins and lipid membranes, as well as the biophysical and cellular determinants of protein misfolding.
