High Resolution Labels for EM

描述（由申请人提供）：EM结构研究的金标签是一种重要工具，可用于进一步了解蛋白质、复合物和细胞组分的组织。荧光标记在光学显微镜水平上非常有用，但对于更高分辨率的EM研究却没有用。随着cryoEM、大阵列CCD、氦载物台、能量过滤器、像差校正器、断层扫描和自动化的出现，电子显微镜最近取得了进展。同样，cryoEM的标本制备方法（规则网格和受控冷冻单元）以及更强大和有用的图像处理软件也取得了重大进展。标记也取得了一些技术进步，例如量子点，ReAsH和小金标签的开发，例如胺和硫醇反应性金簇，FluoroNanogold（结合金和Alexa染料）和Ni-NTA-Nanogold。这些可用于鉴定大分子上的特定位点、复合物中的特定亚基以及细胞中的组分。在这里，我们建议继续开发EM的高分辨率金标签。将建立几个解决当前问题的新标签。这项工作将包括：a）有机合成金壳的专用配体，B）形成稳定且充分表征的金颗粒，c）测试以证明功能性，d）工作以减少或消除非特异性结合，e）分子生物学以引入融合标签，以及e）与合作者一起验证和应用于生物学问题。标签将包括：1. 2-15纳米金标记。功能化的、高度规则的金标记将以更大的尺寸制成，以提高可见性（许多应用所需），并提供反应性金试剂、更大的Ni-NTA-金和改进的金免疫探针。2.金融合标签：功能化以结合蛋白融合标签的金颗粒。a.噬菌体展示标签：噬菌体展示将用于寻找与金标签结合的序列，以用作蛋白质融合标签。连接副本将增加绑定。B.消费税黄金。用GST标记的蛋白质会用金标记这个位点。C. TetraCys金。将制备特异性结合四半胱氨酸基因融合标签的金颗粒。D.钙调素金：钙调蛋白对钙调蛋白结合结构域具有高结合亲和力，并且钙调蛋白-金提供了另一种有用的标签标记替代物。3. PhotoGold具有低结合常数的分子（例如，当它们结合时，金上的肽）将被光锁定，使得能够从未结合的金中纯化并通过EM定位结合位点。4.用于定位ATP和GTP位点的ATP/GTP/AMPPNP/GMPPNP-金。5. DNA嵌入剂金：高度标记的DNA将使其能够在DNA-蛋白质复合物中识别。这些标记将为高分辨率电子显微镜提供重要的新工具。由于缺乏合适的探针，使用电子显微镜进行高分辨率分子分析，这种探针的开发工作将显着推进细胞和分子生物学领域。公共卫生相关性：该项目将通过提供用于在电子显微镜水平上识别分子的新标记，来发现有关重要生物蛋白质、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命过程，这反过来又可以导致疾病的有效治疗。

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.