Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Chemical fluorophores have superior photophysical properties to fluorescent proteins and are much smaller. However, in order to use these probes for live-cell protein imaging, highly specific labeling methods are required. Here, we will describe three efforts to re-engineer the E. coli enzyme, lipoic acid ligase (LplA), to catalyze the ligation of small-molecule probes onto recombinant proteins. We call this collection of methods the PRIME (PRobe Incorporation Mediated by Enzymes) methodologies. First, we describe the structure-guided mutagenesis of LplA and the identification of an LplA variant that can ligate a blue coumarin fluorophore onto a 13-amino acid LplA acceptor peptide (LAP2). This "coumarin ligase" can be used to image cellular proteins with high specificity, sensitivity, and minimal perturbation of the biology of the protein of interest. We also demonstrate how subpopulations of a protein of interest can be labeled using genetically targeted coumarin ligase. Second, we describe our attempts to use yeast display evolution and fluorescence activated cell sorting (FACS) to evolve a truncated LplA enzyme. The original truncated enzyme had severely decreased activity for LplA's natural substrate, lipoic acid. We created a 107 library of LplA mutants and, after four rounds of selection, produced a truncated LplA mutant with lipoylation activity equivalent to full-length LplA. We next sought to evolve activity for an unnatural small molecule probe, but found that this strategy was limited by both increased hydrophobic probe sticking when using the truncated enzyme and some enzyme-dependent nonspecificity. Finally, from a library of 107 LplA mutants, we evolved a full-length LplA capable of ligating an unnatural picolyl azide (pAz) substrate. We demonstrated improved activity of the "pAz ligase" in the secretory pathway and cell surface, two regions where coumarin ligase is inactive. This enzyme can also be used to image cell surface protein-protein interactions as well as label proteins as they are trafficked through the endoplasmic reticulum. These probe ligases will be useful tools for cell biologists interested in studying protein function or protein-protein interactions in the context of living cells. / by Katharine Alice White. / Ph.D.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/78438 |
| Date | January 2012 |
| Creators | White, Katharine Alice |
| 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 | 241 p., application/pdf |
| Rights | M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission., http://dspace.mit.edu/handle/1721.1/7582 |
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