STRUCTURAL AND DYNAMIC ANALYSIS OF PROTEINS IN NEURON CELL MOTILITY

神经元细胞运动中蛋白质的结构和动力学分析

• 批准号: 7960241
• 负责人: BARBARA A LYONS
• 金额: $ 9.65万
• 依托单位: NEW MEXICO STATE UNIVERSITY LAS CRUCES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7960241
• 关键词: Amyloid beta-Protein; Anterior; Behavior; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Models; Biomedical Research; Caenorhabditis elegans; Cells; Classification; Code; Computer Retrieval of Information on Scientific Projects Database; DNA Sequence Rearrangement; Development; Embryo; Embryonic Development; Funding; GTP-Binding Proteins; Genes; Goals; Grant; Institution; Ligands; Location; Measures; Methods; Molecular; Motion; Movement; Mutagenesis; Mutation; Nematoda; Neurons; New Mexico; Nuclear; PH Domain; Phenotype; Phospholipids; Play; Protein Analysis; Protein Binding; Protein Family; Proteins; Relaxation; Research; Research Personnel; Resources; Role; Signal Transduction; Solutions; Source; Structure; System; Tail; Tertiary Protein Structure; Testing; United States National Institutes of Health; Vulva; Wit; Work; cell motility; egg; flexibility; genetic analysis; member; migration; platelet protein P47

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Directed cell migration is essential for spatial ordering of cells during embryogenesis and development. The goal of this research is to determine what role motion and flexibility play in the ability of proteins to recognize and bind specifically to other proteins in cell migration signaling. The chosen model system for this study is the Mig10 protein of the nematode C. elegans. C. elegans is a well characterized system in which the goal of understanding cell migration at the molecular level is attainable. Through genetic analysis the gene Mig10 has been shown to be required for the long range anterior to posterior migration of embryonic neurons and the proper development of the excretory canals. A Mig10 mutation causing severe truncation of the Mig10 protein results in several abnormal phenotypes. The phenotypes include Wit (withered-tail), Egl (egg-laying-defective), Unc (uncoordinated) and protrusion of the vulva. The protein coded by the gene (a.k.a. Mig10) shares a central multidomain region of sequence, termed GM (Grbs and Mig), with the Grb7 protein family. Through homology analysis a Ras-Associating (RA) domain and a Pleckstrin Homology (PH) domain have been identified in the central GM region of the Mig10 and Grb7 family proteins. Adjacent RA and PH domains are also found in other proteins, for example lamellipodin and RIAM (both involved in cytoskeletal rearrangement) and beta-amyloid precursor protein-binding protein, RARP1 (function is unknown). Detailed information describing the function of the Mig10 GM region is not yet existent although some information describing the role of these domains in the Grb7 protein is available. In the Grb7 protein both the PH domain and the RA domain are required for cell migration related to Grb7 signaling. Both the RA and PH domains have eluded easy functional classification, in that the RA domain has not been shown to bind G-proteins, and the PH domain may be a member of a growing group of PH domains that have dual function, binding both phospholipids and proteins. It is possible that the RA and PH domains work in concert, or that prior phospholipid binding is necessary for their activation. Most biochemical methods would be unable to identify function of these domains if that is the case. A combination of structural and dynamic information may the only approach that can elucidate the function of these mysterious domains. The goals of this proposal are: 1) Expression and purification of the Mig10 Pleckstrin Homology and Ras-Associating-like protein domains in quantities sufficient for structure determination, 2) Obtaining the solution structures of both the Mig10-PH and Mig10-RA domains, and 3) Measure the nuclear relaxation behavior of both the PH and RA domains in an effort to relate flexibility and dynamic movement to the location of potential binding sites and ability to bind specific ligands. The accuracy of binding site prediction will then be tested through mutagenesis analysis in C. elegans.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 定向细胞迁移对于胚胎发生和发育过程中细胞的空间有序至关重要。这项研究的目的是确定蛋白质在细胞迁移信号中特异性识别和与其他蛋白质识别和结合的能力中的作用运动和灵活性。这项研究的选定模型系统是线虫C.秀隐杆线虫的MIG10蛋白。秀丽隐杆线虫是一个具有良好特征的系统，在该系统中，可以理解分子水平的细胞迁移的目标。通过遗传分析，基因MIG10已被证明是胚胎神经元前迁移和排泄管的正常发育所必需的。导致MIG10蛋白严重截断的MIG10突变导致几种异常表型。表型包括WIT（枯萎），EGL（卵形缺陷），UNC（不协调）和外阴突出。该基因编码的蛋白质（又称MIG10）与GRB7蛋白质家族共享一个中央序列的中央多域区域，称为GM（GRB和MIG）。通过同源分析，在MIG10和GRB7家族蛋白的中央GM区域已经确定了RAS缔合（RA）结构域和Pleckstrin同源（pH）结构域。在其他蛋白质中也发现了相邻的RA和pH结构域，例如薄片脂蛋白和RIAM（均参与细胞骨架重排）和β-淀粉样蛋白前体蛋白蛋白质结合蛋白，RARP1（功能未知）。描述MIG10 GM区域功能的详细信息尚不存在，尽管一些描述了这些域在GRB7蛋白中的作用的信息。在GRB7蛋白中，pH结构域和RA结构域都是与GRB7信号相关的细胞迁移所必需的。 RA和pH结构域都避免了易于功能分类，因为尚未证明RA结构域结合G蛋白，并且pH结构域可能是具有双重功能的生长型pH结构域的成员，它们具有双重功能，结合了磷脂和蛋白质。 RA和pH结构域可能会协同起作用，或者先前的磷脂结合对于它们的激活是必要的。如果是这种情况，大多数生化方法将无法识别这些域的功能。结构和动态信息的结合可能是唯一可以阐明这些神秘领域功能的方法。该提案的目标是：1）MIG10 PLECKSTRIN同源性和RAS缔合样蛋白结构域的表达和纯化数量足以用于确定结构，2）获得MIG10-PH和MIG10-PH和MIG10-RA结构域的溶液结构，以及3）衡量pH和RA的核能在稳定性中的核能和动态的核能降级，并依赖于稳定性的稳定性和动态稳定性。配体。然后，将通过秀丽隐杆线虫中的诱变分析来测试结合位点预测的准确性。