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THREE DIMENSIONAL STRUCTURE OF ALPHA ACTININ

α 肌动蛋白的三维结构

基本信息

批准号：
6149702
负责人：
KENNETH ALLEN TAYLOR
金额：
$ 20.18万
依托单位：
FLORIDA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-02-01 至 2002-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6149702
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

项目摘要

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-辅肌动蛋白是一种肌动蛋白交联蛋白，在所有真核细胞 α-辅肌动蛋白是最小的成员，血影蛋白-肌养蛋白-α-辅肌动蛋白家族蛋白质。在Z盘中，骨骼肌α-辅肌动蛋白交联反平行肌动蛋白丝，形成I波段。在平滑肌中，α-辅肌动蛋白位于粘附斑块和致密体。在组织培养的细胞中，α-辅肌动蛋白是位于粘连灶和应力纤维中，粘连的结构类似物斑块和致密体。 α-辅肌动蛋白中的分子缺陷被认为导致某些形式的肌肉疾病线虫肌病，而缺陷，与之密切相关的抗肌萎缩蛋白分子导致杜氏肌营养不良症营养不良只有很少的三维结构信息可用于这些分子。该项目的工作是第一步，长期目标是研究复杂的分子相互作用，发生在Z盘、粘连斑块和局灶性粘连以及其他部位，肌动蛋白丝用于形成肌肉和细胞骨架结构。博士泰勒和他的同事最近表明，光滑的二维晶体适合于电子晶体学的肌肉α-辅肌动蛋白可以在带正电荷的脂质单层。这是首次报道的 α-辅肌动蛋白的结晶，从而促进三维结构保持战略定力有人建议利用这一进步，并利用这些晶体来确定α-辅肌动蛋白的结构，具体目标如下：（1）产生平滑肌的3D图像 α-辅肌动蛋白来验证所建议的蛋白质结构域的比对。（二）产生平滑肌α辅肌动蛋白和当与肌动蛋白结合时，肌动蛋白决定方向。 (3)结晶阿米巴α-辅肌动蛋白，并获得一个图像，揭示了对齐蛋白质结构域。 (4)检查肌动蛋白丝交叉的方向与阿米巴α辅肌动蛋白有关。 (5)确定条件，两者都产生一种或多种α-辅肌动蛋白同种型的晶体，适用于原子结构分析并保存其晶体在电子显微镜下。