Structure and Conformational Changes of Assembled Dynamin

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DESCRIPTION (provided by applicant): Dynamin is a large, multidomain GTPase essential for the final steps of clathrin mediated endocytosis and is a key regulator of synaptic vesicle recycling. In order to understand the mechanisms by which it modulates these biological events, it is crucial to determine how dynamin translates nucleotide hydrolysis into structural changes to facilitate membrane fission and the liberation of clathrin coated vesicles into the cell. Despite large amounts of functional data, the exact nature of the conformational changes that drive dynamin's mechanochemical activities is still ill-defined due to a lack of structural information. To better understand how dynamin structure influences its functional activities, we seek to visualize the structure and conformational changes of assembled dynamin using a combination of cryo-electron microscopy, biochemistry, and image reconstruction techniques. These studies will yield three-dimensional reconstructions of dynamin at sufficient resolution to allow us to see the key structural changes that are linked to different nucleotide states. Attachment of electron-dense labels will elucidate the overall organization of the dynamin polymer and will unambiguously define the positions of dynamin's domains within these structural maps. This knowledge will greatly increase our understanding of how dynamin transmits the chemical energy of GTP hydrolysis into the physical work of membrane scission and ultimately pre-synaptic recycling. The ability of the body to transmit neurological impulses is highly dependent on the efficiency of a process known as pre-synaptic recycling, which maintains a large pool of vesicles pre-loaded with neurotransmitters and primes the neuron for rapid response to an action potential. The GTPase dynamin is essential for this process, though the underlying mechanisms of its function remain unknown due to a lack of structural information about the protein. We seek to determine how dynamin structure influences its function, thereby establishing the key changes in the protein that drive its biological activities. These findings will ultimately enhance our understanding of dynamin's role in mental health.