Over the past decade chemical tags have been developed to complement the use of fluorescent proteins in live cell imaging. Chemical tags retain the specificity of protein labeling achieved with fluorescent proteins through genetic encoding, but provide smaller, more robust tags and modular use of organic fluorophores with high photon-output and tailored functionalities. The trimethoprim-based chemical tag (TMP-tag) was initially developed based on the high affinity interaction between E.coli dihydrofolatereductase and the antibiotic trimethoprim and subsequently rendered covalent and fluorogenic via proximity-induced protein labeling reactions. To date, the TMP-tag is one of the few chemical tags that enable intracellular protein labeling and high-resolution live cell imaging. In this dissertation I first focused on the development of the fluorogenic TMP-tag that enabled high resolution live cell imaging with redeced background noise. Then some efforts to further improve the fluorogenic TMP-tag were described. Furthermore, I worked with collaborators to apply the TMP-tag technology to study the dynamics of the focal adhesion complex at the single-molecule level. Finally, I also tried to develop a short peptide tag for protein labeling with minimal perturbation of the function and dynamics of the target molecule. Together, these studies exemplified the maturation of the TMP-tag technology from the proof-of-principle stage to real-world biological applications.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/D8639P5V |
| Date | January 2013 |
| Creators | Jing, Chaoran |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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