title: Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein.
abstract: The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 Å resolution. The hexagonal-shaped crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 Å, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis.
authors.affiliation: Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria.
authors.forename: Eva
authors.initials: E
authors.lastname: Klinglmayr
authors.forename: Julia
authors.initials: J
authors.lastname: Wenger
authors.forename: Sandra
authors.initials: S
authors.lastname: Mayr
authors.forename: Ella
authors.initials: E
authors.lastname: Bossy-Wetzel
authors.forename: Sandra
authors.initials: S
authors.lastname: Puehringer
mesh_terms: D003460:Crystallization; D018360:Crystallography, X-Ray; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000302:isolation & purification; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D008869:Microtubule-Associated Proteins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D020558:GTP Phosphohydrolases; C110768:DNM1L protein, human; D034281:Dynamins