Working Towards Cryo-Em Structures of Mitochondrial Dynamin-Like GTPases

Abstract

Dynamins are a class of GTPase enzymes responsible for the fusion, fission, and vesiculation of cellular lipid membranes throughout the cell. The dynamin-like proteins Mitofusin (Mfn1/2) and Optic Atrophy 1 (Opa1) are responsible for the fusion of the outer and inner mitochondrial membranes, respectively. Unlike other dynamin-like proteins, Mfn1/2 and Opa1 are transmembrane proteins. Currently, structural data for Mfn1/2 in association with membranes is limited and any structural or biochemical data for Opa1 is even more so. We have developed protocols for systematically expressing Mfn1/2 and are working towards purification in a lipid bilayer. Additionally, expression and purification of biologically relevant and biochemically active shortened isoforms of Opa1 in sufficient quantity to conduct biochemical assays have also been achieved. Furthermore, we are optimizing sample preparation and have begun reconstructions of a proteolytically processed short form, s-Opa1, by cryo-EM on a lipid surface and are examining the role nucleotide states play in structural rearrangements.