THREE DIMENSIONAL STRUCTURE OF ALPHA ACTININ

α 肌动蛋白的三维结构

• 批准号: 2653905
• 负责人: KENNETH ALLEN TAYLOR
• 金额: $ 18.51万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-02-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2653905
• 关键词: Acanthamoeba; alpha actinin; crystallins; crystallization; cytoskeletal proteins; electron microscopy; laboratory rabbit; muscle proteins; protein isoforms; protein structure; smooth muscle; structural biology; tomography

DESCRIPTION: Alpha-actinin is an actin cross linking protein found in all eucaryotic cells. Alpha-actinin is the smallest member of the spectrin-dystrophin-alpha-actinin family of proteins. In the Z-disk of skeletal muscle alpha-actinin cross links antiparallel actin filaments to form the I-band. In smooth muscle alpha-actinin is located in adhesion plaques and dense bodies. In tissue cultured cells, alpha-actinin is located in focal adhesions and stress fibers, structural analogs of adhesion plaques and dense bodies. Molecular defects in alpha-actinin are thought to cause some forms of the muscle disease nemaline myopathy, while defects in the closely related molecule dystrophin cause the disease Duchenne muscular dystrophy. There is very little 3-D structural information available for these molecules. The work of this project is the first step in the long-range goal of investigating the complex molecular interactions that occur in Z-disks, adhesion plaques and focal adhesions and other sites where actin filaments are used to form muscle and cytoskeletal structures. Dr. Taylor and colleagues have recently shown that 2-D crystals of smooth muscle alpha-actinin suitable for electron crystallography can be formed on positively charged lipid monolayers. This is the first reported crystallization of alpha-actinin thereby facilitating a 3-D structure determination. It is proposed to exploit this advance and use these crystals to determine the structure of alpha-actinin according to the following specific aims: (1) Produce a 3-D image of the smooth muscle alpha-actinin to verify the suggested alignment of protein domains. (2) produce a 3-D image of the complex between smooth muscle alpha actinin and actin to determine the orientation when bound to actin. (3) Crystallize Acanthamoeba alpha-actinin and obtain an image that reveals the alignment of protein domains. (4) Examine the orientation of actin filaments cross linked with Acanthamoeba alpha-actinin. (5) Determine conditions that will both produce crystals of one or more alpha-actinin isoforms of a size suitable for an atomic structure analysis and preserve their crystalline order in the electron microscope.

描述：α-肌动蛋白是一种肌动蛋白交联蛋白，存在于 真核细胞。α-肌动蛋白是最小的成员 血影蛋白-肌营养不良蛋白-α-肌动蛋白家族。在的Z圆盘中 骨骼肌α-肌动蛋白将反平行肌动蛋白细丝与 组成I-Band。在平滑肌中，α-肌动蛋白定位于粘连。 斑块和致密的身体。在组织培养细胞中，α-肌动蛋白是 定位于局灶性粘连和应力纤维，粘连的结构类似物 斑块和致密的身体。α-肌动蛋白的分子缺陷被认为是 导致某些形式的肌肉疾病线状肌病，而缺陷 与Duchenne肌肉疾病密切相关的分子Dstrophin 营养不良。几乎没有什么三维结构信息可用于 这些分子。这一项目的工作是 长期目标是研究复杂的分子相互作用 发生在Z型盘、粘连斑块和灶性粘连等部位 肌动蛋白细丝用于形成肌肉和细胞骨架结构。 泰勒博士和他的同事最近证明了光滑的二维晶体 适合于电子结晶学的肌肉α-肌动蛋白可以在 带正电的脂单分子层。这是首次报道 α-肌动蛋白的结晶，从而促进三维结构 决心。建议利用这一进步并使用这些 晶体来确定α-肌动蛋白的结构 具体目标如下：(1)制作平滑肌肉的3-D图像 α-肌动蛋白来验证所建议的蛋白质结构域的比对。(2) 制作平滑肌α-肌动蛋白和肌动蛋白复合体的三维图像 肌动蛋白用于确定与肌动蛋白结合时的方向。(3)结晶 棘阿米巴α-肌动蛋白，并获得一张揭示了 蛋白质结构域。(4)观察肌动蛋白细丝交叉的方向。 与棘阿米巴α-肌动蛋白相连。(5)确定将 两者都产生一种或多种大小相同的α-肌动蛋白亚型的晶体。 适用于原子结构分析和保存它们的晶体 在电子显微镜下进行排序。