High Resolution Labels for EM

EM 高分辨率标签

• 批准号: 7655799
• 负责人: James F Hainfeld
• 金额: $ 37.36万
• 依托单位: NANOPROBES, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7655799
• 关键词: Address; Affinity; Amines; Automation; Binding; Binding Sites; Biological; Calmodulin; Cells; Chimeric Proteins; Complex; Computer software; Cryoelectron Microscopy; Cysteine; DNA; Development; Disease; Docking; Dyes; Electron Microscope; Electron Microscopy; Freezing; Genetic; Gold; Guanosine Triphosphate; Helium; Intercalating Agents; Label; Lead; Life; Ligands; Light Microscope; Methods; Molecular Analysis; Molecular Biology; Molecular and Cellular Biology; Organic Synthesis; Peptides; Phage Display; Preparation; Process; Proteins; Quantum Dots; Reagent; Resolution; Site; Specimen; Spiders; Staging; Sulfhydryl Compounds; Testing; Validation; Work; effective therapy; image processing; improved; macromolecule; nanoGold; particle; protein complex; public health relevance; tomography; tool

DESCRIPTION (provided by applicant): Gold labels for EM structural studies are an important tool that can be used to further understand the organization of proteins, complexes and cellular components. Fluorescent labels have been incredibly useful at the light microscope level, but are not useful for higher resolution EM studies. Electron microscopes have recently advanced with the advent of cryoEM, large array CCDs, helium stages, energy filters, aberration correctors, tomography, and automation. Similarly, significant advances have been made in specimen preparation methods for cryoEM (regular grids and controlled freezing units) and more powerful and useful image processing software. Labeling has also seen some technological advances, such as the development of quantum dots, ReAsH, and small gold labels, such as amine and thiol reactive gold clusters, FluoroNanogold (combining gold and Alexa dyes) and Ni-NTA-Nanogold. These are useful for identifying specific sites on macromolecules, particular subunits in a complex, and components in cells. Here we propose to continue development of high resolution gold labels for EM. Several new labels addressing current problems will be constructed. The work will include: a) organic synthesis of specialized ligands for the gold shell, b) formation of stable and well-characterized gold particles, c) testing to demonstrate functionality, d) work to reduce or eliminate non-specific binding, e) molecular biology to introduce fusion tags, and e) validation and application to biological problems with collaborators. Label will include: 1. 2-15 nm gold labels. Functionalized, highly regular, gold labels will be made in larger sizes to improve visibility (needed for many applications) and provide reactive gold reagents, larger Ni-NTA-gold, and improved gold immunoprobes. 2. Gold Fusion-Tag Labels: Gold particles functionalized to bind to protein fusion tags. a. Phage Display Tags: Phage display will be used to find sequences that bind to gold labels for use as protein fusion tags. Concatenated copies will increase binding. b. GST gold. Proteins tagged with GST would have this site labeled with gold. c. TetraCys gold. Gold particles will be made that specifically bind to the tetra-cysteine genetic fusion tag. d. Calmodulin gold: Calmodulin has a high binding affinity for the calmodulin binding domain, and calmodulin-gold provides another useful tag labeling alternative. 3. PhotoGold. Molecules with low binding constants (e.g., peptides) on the gold will be photolocked when they bind, enabling purification from unbound gold and binding site localization by EM. 4. ATP/GTP/AMPPNP/GMPPNP-gold for localizing ATP and GTP sites. 5. DNA intercalator gold: Highly labeled DNA will enable its identification in DNA-protein complexes. This arsenal of labels will provide important new tools for high resolution electron microscopy. Given the paucity of suitable probes for high resolution molecular analysis using electron microscopy, this probe development work would significantly advance the fields of cell and molecular biology. PUBLIC HEALTH RELEVANCE: This project will enable important new information to be found about important biological proteins, complexes, and cells by providing new labels for identifying molecules at the electron microscope level. The ultimate aim of these studies is to understand life processes, which in turn, can lead to effective treatment of disease.

描述（由申请人提供）：用于EM结构研究的金标签是一个重要的工具，可用于进一步了解蛋白质，复合物和细胞成分的组织。荧光标签在光学显微镜水平上非常有用，但对于高分辨率EM研究并不有用。电子显微镜最近随着冷冻，大阵列CCD，氦气阶段，能量过滤器，畸变校正器，断层扫描和自动化的出现。同样，在冷冻的标本准备方法（常规网格和受控冻结单元）和更强大，更有用的图像处理软件中也取得了重大进展。标签还看到了一些技术进步，例如量子点的发展，reash和小金标签，例如胺和硫醇反应性金簇，氟纳米元（结合金和Alexa染料）和Ni-Nta-Nta-Nanogold。这些对于鉴定大分子，复合物中的特定亚基以及细胞中的成分很有用。在这里，我们建议继续开发EM的高分辨率黄金标签。将构建一些解决当前问题的新标签。这项工作将包括：a）用于黄金外壳的特殊配体的有机合成，b）形成稳定且特征良好的金颗粒，c）测试以证明功能，d）减少或消除非特异性结合的工作，e）分子生物学，以引入融合和对生物学问题的验证和应用于生物学问题。标签将包括：1。2-15nm金标签。功能化，高度规则的黄金标签将以较大尺寸制成，以提高可见性（许多应用所需），并提供反应性的金试剂，较大的Ni-NTA金和改进的金免疫探针。 2。金融合标签标签：功能化与蛋白质融合标签结合的金颗粒。一个。噬菌体显示标签：噬菌体显示将用于查找与金标签结合的序列，以用作蛋白质融合标签。串联副本将增加结合。 b。 GST黄金。用GST标记的蛋白质将其标记为黄金。 c。 tetracys黄金。将制作出特异性结合四型甲基遗传融合标签的金颗粒。 d。钙调蛋白黄金：钙调蛋白对钙调蛋白结合域具有很高的结合亲和力，而钙调蛋白 - 金可提供另一种有用的标签标签替代品。 3。摄影。具有低结合常数（例如，肽）的分子在结合时将被光锁定，从而从未结合的黄金和结合位点的定位中纯化。 4。用于本地化ATP和GTP位点的ATP/GTP/AMPPNP/GMPPNP金。 5。DNA介导子金：高标记的DNA将使其在DNA蛋白复合物中的鉴定。这种标签的武器库将为高分辨率电子显微镜提供重要的新工具。考虑到使用电子显微镜进行高分辨率分子分析的合适探针的匮乏，该探针开发工作将显着推进细胞和分子生物学的领域。公共卫生相关性：该项目将通过提供新的标签来识别电子显微镜水平的分子，从而使有关重要的生物蛋白，复合物和细胞的重要新信息。这些研究的最终目的是了解生活过程，这反过来又可以导致对疾病的有效治疗。