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.
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.
|Joshi, V N; Mitra, D; England, M D et al. (2010) Large Covalently Linked Fluorescent and Gold Nanoparticle Immunoprobes. Microsc Microanal 16:966-967|
|Ackerson, Christopher J; Powell, Richard D; Hainfeld, James F (2010) Site-specific biomolecule labeling with gold clusters. Methods Enzymol 481:195-230|