Expression and Characterization of Torsin A

肌张力障碍是一种神经系统疾病，表现为不受控制的持续肌肉收缩。 早发性肌张力障碍发生在儿童和青少年。 早发型肌张力障碍的遗传位点在1997年被确定。 该基因座内含有扭转蛋白A基因。 研究表明，所有患有早发性肌张力障碍的个体在该基因中都有一处损伤，即编码区3'端附近的一个谷氨酸残基丢失。目前，TorsinA在正常和受影响个体中的功能尚不清楚。扭转蛋白A的结构信息在确定其在正常神经功能中的作用方面是非常宝贵的。 此外，确定Glu缺失的后果将提供对突变蛋白的异常功能的了解，并可能有助于确定对早发性肌张力障碍的治疗。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。 这一目标分为若干不同的阶段： o获得高水平的TorsinA生物表达 〇确定扭转蛋白A的溶液和/或晶体结构 o研究由于Glu 302缺失而导致的扭转蛋白A的结构变化 o研究其他药物对神经功能的影响 扭转蛋白A中的突变在R21建议的过程中，我们计划在细菌、酵母或哺乳动物系统中获得扭转蛋白A的高水平表达。一旦鉴定出合适的扭转蛋白A来源，将使用标准生物化学技术获得纯蛋白。 将评估该材料通过NMR光谱或X射线衍射进行结构测定的适用性。 如果获得晶体，我们将开始用晶体学测定结构。 如果晶体生长有问题，我们将优化溶液条件，通过NMR光谱进行结构测定。 此外，我们还将继续研究扭转蛋白A的生化特性，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.