Expression and Characterization of Torsin A

肌张力障碍是一种神经系统疾病，表现为无法控制和持续的肌肉收缩。早发性肌张力障碍发生于儿童和青少年。早发性肌张力障碍的基因位点于1997年确定。在这个基因座中包含了TorsinA基因。研究表明，所有患有早发性肌张力障碍的个体都有该基因的单一病变；在编码区3'端附近丢失一个谷氨酸残基。目前TorsinA在正常和受影响个体中的功能尚不清楚。TorsinA的结构信息对于确定其在正常神经功能中的作用是非常宝贵的。此外，确定Glu缺失的后果将有助于深入了解nutant蛋白的异常功能，并可能有助于确定早发性肌张力障碍的治疗方法。本研究计划的长期目标是确定TorsinA的结构与其在肌张力障碍中的作用之间的关系。这一目标分为几个不同的阶段：获得TorsinA生物体的高水平表达，确定TorsinA的溶液和/或晶体结构，研究由于Glu302缺失导致的TorsinA结构变化，研究TorsinA其他突变对神经功能的影响。在R21提案的过程中，我们计划在细菌，酵母或哺乳动物系统中获得TorsinA的高水平表达。一旦确定了合适的TorsinA来源，将使用标准的生化技术获得纯蛋白。将评估该材料用于核磁共振光谱复制或X射线衍射测定结构的适用性。如果获得晶体，我们将开始用晶体地理学测定结构。在晶体生长有问题的情况下，我们将优化溶液条件，用核磁共振波谱法确定结构。此外，我们还将对TorsinA进行生化表征，以确定其在神经功能中的作用。

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.