title: Recombinant expression, purification, and comparative characterization of torsinA and its torsion dystonia-associated variant Delta E-torsinA.
abstract: Early-onset torsion dystonia is an autosomal dominant movement disorder that has been linked to the deletion of one of a pair of glutamic acid residues in the protein torsinA (E(302/303); DeltaE-torsinA). In transfected cells, DeltaE-torsinA exhibits similar biochemical properties to wild type (WT)-torsinA, but displays a distinct subcellular localization. Primary structural analysis of torsinA suggests that this protein is a membrane-associated member of the AAA family of ATP-binding proteins. However, to date, neither WT- nor DeltaE-torsinA has been obtained in sufficient quantity and purity to permit detailed biochemical and biophysical characterization. Here, we report a baculovirus expression system that provides milligram quantities of purified torsin proteins. Recombinant WT- and DeltaE-torsinA were found to be membrane-associated glycoproteins that required detergents for solubilization and purification. Analysis of the biophysical properties of WT- and DeltaE-torsinA indicated that both proteins were folded monomers in solution that exhibited equivalent denaturation behaviors under thermal and chaotropic (guanidinium chloride) stress. Additionally, both forms of torsinA were found to display ATPase activity with similar k(cat) and K(m) values. Collectively, these data reveal that torsinA is a membrane-associated ATPase and indicate that the DeltaE(302/303) dystonia-associated mutation in this protein does not cause gross changes in its catalytic or structural properties. These findings are consistent with a disease mechanism in which DeltaE-torsinA promotes dystonia through a gain rather than loss of function. The recombinant expression system for torsinA proteins described herein should facilitate further biochemical and structural investigations to test this hypothesis.
authors.affiliation: The Skaggs Institute for Chemical Biology and Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Kustedjo
authors.forename: Songpon
authors.initials: S
authors.lastname: Deechongkit
authors.forename: Jeffery W
authors.initials: JW
authors.lastname: Kelly
authors.forename: Benjamin F
authors.initials: BF
authors.lastname: Cravatt
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D002460:Cell Line; D002942:Circular Dichroism; D004422:Dystonia Musculorum Deformans / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D005822:Genetic Vectors; D019791:Guanidine; D006801:Humans; D018832:Molecular Chaperones / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D018411:Spodoptera; D013816:Thermodynamics; D014461:Ultracentrifugation
chemical_list: D002352:Carrier Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; C108175:TOR1A protein, human; D000251:Adenosine Triphosphatases; D019791:Guanidine