STRUCTURE AND FUNCTION OF A CRYSTALLINE ACTIN BUNDLE

结晶肌动蛋白束的结构和功能

• 批准号: 2191822
• 负责人: PAUL T. MATSUDAIRA
• 金额: $ 21.44万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2191822
• 关键词: SDS polyacrylamide gel electrophoresis; acrosome; actin binding protein; actins; antibody; chemical binding; chemical fingerprinting; crosslink; cytoskeletal proteins; cytoskeleton; electron microscopy; high performance liquid chromatography; horseshoe crabs; immunofluorescence technique; laboratory rabbit; mass spectrometry; molecular cloning; polymerase chain reaction; protein biosynthesis; protein sequence; protein structure function; stoichiometry; western blottings

Stiff bundles of actin filaments provide structural support for fingers of membrane such as microvilli, stereocilia, and filopodia. One model system for studying the structure, function, and assembly of actin bundles is the crystalline bundle in the acrosomal process of Limulus sperm. In unactivated sperm, the bundle is coiled around the base of the nucleus but when the sperm contacts the egg, the actin bundle straightens and extends to its full 80 mum length in a few minutes. The acrosomal process is a simple structure, composed of actin and the actin crosslinking protein scruin, a 102 kDa polypeptide complexed with two 17 kDa polypeptides. The 13 A resolution 3D structure of a filament in the acrosomal process scruin is organized into two domains which bind to two actin subunits. The sequence of the 102 kDa polypeptide is divided into a tandem pair of homologous domains each containing a six-fold repeat of a 50 residue sequence. A search of the structure database reveals this sequence corresponds to a four-stranded beta-sheep motif which are repeated five, six, or seven times for form a disc-shaped beta-flower domain. This domain is found in viruses, procaryotes, and eucaryotes. In addition to scruin, the beta-flower domain is also found in Drosophila ovary kelch, C. elegans sperm spe26, and mouse placental MIPP. Because these proteins have not been isolated, their functions have not been studied but their similarity with scruin suggests a cytoskeletal function. Our goal is to describe how scruin crosslinks actin filaments into a bundle. To accomplish this goal we have two specific aims. The first aim is to describe completely the structure and function of scruin. Biochemical and molecular biology experiments will describe the organization of scruin domains, their association with the 17 kDa polypeptide, the binding sites on actin, and the actin binding sites on scruin. The second goal is to determine the 3D structure of the actin bundle in collaboration with the cryoEM group at Baylor. We will provide them with actin filaments decorated with scruin domains, specific anti- scruin antibodies, and scruin labelled at specific residues with gold clusters. Using the technique of image reconstruction from cryoelectron micrographs, the Baylor group will map the location of residues in the sequence to the 3D structure. This information will be crucial to later studies to determine the structure of the filament to a resolution of 6A. Because scruin and its related proteins are involved in some aspect of gametogenesis, our studies may be relevant to studies in human reproduction and early development.

肌动蛋白丝的僵硬捆为手指的结构支撑 膜，例如微绒毛，立体尾膜和丝状虫。 一个模型系统 用于研究肌动蛋白束的结构，功能和组装是 晶体捆绑包在Limulus精子的教科体过程中。 在 未活化的精子，将束缠绕在细胞核的底部，但 当精子接触卵时，肌动蛋白束会拉直并延伸 在几分钟内完成整个80妈妈的长度。 杂点过程是 简单结构，由肌动蛋白和肌动蛋白交联蛋白组成 Scruin，一种102 kDa多肽，与两个17 kDa多肽复合。 这 13 AROSOMAL工艺过程中细丝的分辨率3D结构 被组织成两个结合到两个肌动蛋白亚基的结构域。 这 102 kDa多肽的序列分为一对串联 同源域，每个域都包含50个残基的六倍重复 顺序。 对结构数据库的搜索显示了此序列 对应于四链β-肩主题，重复五个， 形成圆盘形的β-花域的六次或七次。 该结构域在病毒，丙烯酸酯和桉树中发现。 此外 对于Scruin，在果蝇卵巢Kelch中也发现了β-花域， C.秀丽隐杆线虫精子SPE26和小鼠胎盘Mipp。 因为这些蛋白质 尚未孤立，他们的功能尚未被研究，但他们的功能 与SCRUIN的相似性表明细胞骨架功能。 我们的目标是描述Scruin如何将肌动蛋白丝交联 捆。 为了实现这一目标，我们有两个具体的目标。 第一个目标 是完全描述Scruin的结构和功能。 生化和分子生物学实验将描述 Scruin域的组织，它们与17 kDa的关联 多肽，肌动蛋白上的结合位点以及肌动蛋白结合位点 Scruin。 第二个目标是确定肌动蛋白的3D结构 与贝勒的冷冻小组合作的捆绑包。 我们将提供 它们用肌动蛋白丝装饰有scuin域，特定的抗 Scruin抗体，并在特定残基上标记为黄金 集群。 使用Cryoelectron的图像重建技术 显微照片，贝勒组将绘制残留物的位置 3D结构的序列。 此信息对于以后至关重要 研究丝的结构为6a的分辨率。 因为Scruin及其相关蛋白在某些方面涉及 配子发生，我们的研究可能与人类繁殖研究有关 和早期发展。