Expression and Characterization of Torsin A

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Dystonia is a neurological disease that presents as uncontrolled and sustained muscle contractions. Early onset dystonia occurs in childhood and adolescents. The genetic locus responsible for early onset dystonia was identified in 1997. Contained within this locus is the gene for TorsinA. It has been shown that all individuals possessing early onset dystonia have a single lesion in this gene; the loss of a single Glutamic acid residue near the 3' end of the coding region. Currently the function of TorsinA in normal and affected individuals is unknown. Structural information on TorsinA would be invaluable in defining it role in normal neurological function. In addition, determining the consequences of the Glu deletion will provide insight into the aberrant function of the nutant protein an may aid in defining a treatment for early onset dystonia. The long-term goal of this research plan is to determine the relationship between the structure of TorsinA and its role in dystonia. This goal is divided into a number of distinct phases: o Obtain high levels of expression of TorsinA organisms o Determine the solution and/or crystal structure of TorsinA o Investigate structural changes in TorsinA due to deletion of Glu302 o Investigate the effect on neurological function of other mutations in TorsinA During the course of this R21 proposal we plan on obtaining high-level of expression of TorsinA in bacterial, yeast, or mammalian systems. Once a suitable source of TorsinA is identified, pure protein will be obtained using standard biochemical techniques. The suitability of this material for structure determination by either NMR spectros-copy or X- ray diffraction will be assessed. If crystals are obtained we will initiate structure determination by crystal-lography. In the event that crystal growth is problematic we will optimize solution conditions for structure determination by NMR spectroscopy. In addition, we will also pursue biochemical characterization of TorsinA to determine its role in neurological function.