Expression and Characterization of Torsin A

Torsin A 的表达和表征

• 批准号: 6361757
• 负责人: GORDON S. RULE
• 金额: $ 10.82万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-15 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6361757
• 关键词: Escherichia coli; Mammalia; X ray crystallography; adenosine triphosphate; binding proteins; computer simulation; crystallization; dystonia; gel filtration chromatography; gene mutation; glutamates; ion exchange chromatography; model design /development; molecular pathology; nerve /myelin protein; neuropathology; nuclear magnetic resonance spectroscopy; physical model; protein purification; protein structure function; structural biology; technology /technique development; transfection /expression vector; western blottings; yeasts

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.

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于儿童期和青少年。 导致早发性肌张力障碍的基因位点于 1997 年被发现。该位点内包含 TorsinA 基因。 研究表明，所有早发性肌张力障碍患者的该基因均存在单一损伤。编码区 3' 端附近单个谷氨酸残基的丢失。目前 TorsinA 在正常人和受影响个体中的功能尚不清楚。 TorsinA 的结构信息对于定义它在正常神经功能中的作用非常有价值。 此外，确定 Glu 缺失的后果将有助于深入了解坚果蛋白的异常功能，并可能有助于确定早发性肌张力障碍的治疗方法。该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。 该目标分为多个不同的阶段： o 获得 TorsinA 生物体的高水平表达 o 确定 TorsinA 的溶液和/或晶体结构 o 研究由于 Glu302 缺失而导致的 TorsinA 结构变化 o 研究 Glu302 缺失对 TorsinA 神经功能的影响TorsinA 中的其他突变 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 TorsinA 的合适来源，将使用标准生化技术获得纯蛋白质。 将评估该材料通过 NMR 光谱或 X 射线衍射确定结构的适用性。 如果获得晶体，我们将通过晶体学开始结构测定。 如果晶体生长出现问题，我们将优化溶液条件，通过核磁共振波谱确定结构。 此外，我们还将研究 TorsinA 的生化特征，以确定其在神经功能中的作用。