High Resolution Labels for EM

描述(申请人提供)：EM结构研究的金标是一个重要的工具，可以用来进一步了解蛋白质、复合体和细胞组件的组织。荧光标记在光学显微镜水平上非常有用，但在更高分辨率的EM研究中用处不大。近年来，随着低温电子显微镜、大阵列电荷耦合器件、氦气台、能量滤光器、像差校正器、层析成像和自动化的出现，电子显微镜得到了发展。同样，在低温电子显微镜(规则网格和受控冷冻单元)的样品制备方法以及更强大和更有用的图像处理软件方面也取得了重大进展。标记也取得了一些技术进步，如量子点、ReAsH和小金标的开发，如胺和硫醇活性金簇、荧光纳米金(结合金和Alexa染料)和镍-NTA-纳米金。这些有助于识别大分子上的特定位置、复合体中的特定亚基和细胞中的成分。在这里，我们建议继续开发用于EM的高分辨率金标签。将建造几个新的标签来解决当前的问题。这项工作将包括：a)用于金壳的专门配体的有机合成，b)形成稳定和特征良好的金粒子，c)测试以证明功能性，d)努力减少或消除非特异性结合，e)分子生物学引入融合标签，e)与合作者验证和应用于生物学问题。标签将包括：1.2-15纳米黄金标签。功能化、高度规则的金标签将制成更大尺寸的标签，以提高可见性(许多应用都需要)，并提供活性金试剂、更大的镍-NTA-金和改进的金免疫探针。2.金融合标签：功能化的金颗粒与蛋白质融合标签结合。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。串联的副本将增加约束力。B.商品及服务税黄金。标记了GST的蛋白质会将这个位置标记为金。C.TetraCys黄金。将制造出与四半胱氨酸基因融合标签特异结合的金粒子。D.钙调蛋白金：钙调蛋白与钙调蛋白结合域具有很高的结合亲和力，钙调素-金提供了另一种有用的标记替代方法。3.PhotoGold。在金表面具有低结合常数的分子(例如，多肽)在结合时将被光锁定，从而能够从未结合的金中提纯，并通过EM定位结合部位。4.用于定位ATP和GTP站点的ATP/GTP/AMPPNP/GMPPNP-GOLD。5.DNA嵌入剂金色：高标记的DNA将使其能够在DNA-蛋白质复合体中识别。这一标记库将为高分辨率电子显微镜提供重要的新工具。鉴于用于电子显微镜高分辨率分子分析的合适探针的匮乏，这项探针开发工作将极大地推动细胞和分子生物学领域的发展。与公共卫生相关：该项目将通过提供在电子显微镜水平上识别分子的新标记，使人们能够找到关于重要生物蛋白质、复合体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，这反过来可以导致有效的疾病治疗。

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.