Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Protein interaction networks and protein compartmentalization underlie all signaling and regulatory processes in cells. Traditional approaches to proteomics employ mass spectrometry (MS) coupled to biochemical fractionation or affinity purification but require cell lysis prior to analysis which often results in false-negatives from missed interactions or incomplete purification and false-positives from contaminants. Enzyme-catalyzed proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in which a PL enzyme can be genetically targeted to a subcellular region and used to tag surrounding endogenous proteins with a chemical handle that allows their identification by MS. Tagging is carried out in living cells in a distance-dependent manner, allowing data collection from a physiologically relevant environment with preservation of spatial information. Current PL methods are limited by poor catalytic efficiency or toxic substrates that limit their application in vivo. Therefore, we have developed a new proximity labeling method, called TurboID, that uses non-toxic labeling conditions and has high catalytic efficiency that allows its use in a wide variety of biological contexts. Here, we describe our use of yeast display-based directed evolution to engineer two promiscuous mutants of biotin ligase, TurbolD and miniTurbo. We describe our characterization of the evolved PL enzymes in microbes, cultured cells, in vitro, and in vivo in flies and worms, and show that TurbolD and miniTurbo have much greater catalytic efficiency than any other biotin ligase-based PL method currently available. Lastly, we demonstrate that TurbolD and miniTurbo can be used to obtain proteomes with the same size, specificity, and depth-of-coverage as existing biotin-ligase based PL techniques with over 100- fold shorter labeling times. In the Appendix, we discuss two separate projects. In Part I, we describe how fusion of the PL enzyme APEX2 to various mitochondrial proteins could be used to map the proteomes of mitochondrial subdomains and be used to visualize the localization of mitochondrial proteins in mitochondrial subdomains using APEX2 to generate contrast for electron microscopy imaging. In Part II, we discuss the development of two platforms that could be used to temporally control genome editing using light. / by Tess C. Branon. / Ph. D.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/120909 |
| Date | January 2018 |
| Creators | Branon, Tess C |
| Contributors | Alice Y. Ting., Massachusetts Institute of Technology. Department of Chemistry., Massachusetts Institute of Technology. Department of Chemistry. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 167 pages, application/pdf |
| Rights | MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission., http://dspace.mit.edu/handle/1721.1/7582 |
Page generated in 0.0022 seconds