New reporters of protein trafficking and protein-protein interactions in live cells
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | eng |
| Published: |
Massachusetts Institute of Technology
2009
|
| Subjects: | |
| Online Access: | http://hdl.handle.net/1721.1/44678 |
| _version_ | 1826204005676613632 |
|---|---|
| author | Fernández Suárez, Marta |
| author2 | Alice Y. Ting. |
| author_facet | Alice Y. Ting. Fernández Suárez, Marta |
| author_sort | Fernández Suárez, Marta |
| collection | MIT |
| description | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008. |
| first_indexed | 2024-09-23T12:47:19Z |
| format | Thesis |
| id | mit-1721.1/44678 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T12:47:19Z |
| publishDate | 2009 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/446782019-04-11T10:30:13Z New reporters of protein trafficking and protein-protein interactions in live cells Fernández Suárez, Marta Alice Y. Ting. Massachusetts Institute of Technology. Dept. of Chemistry. Massachusetts Institute of Technology. Dept. of Chemistry. Chemistry. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008. Vita. Includes bibliographical references. Here, we describe our attempts to harness the exquisite specificity of natural protein and RNA enzymes to develop improved methods to study protein localization and protein-protein interactions in live cells. We first attempted to detect endogenous protein-protein interactions (PPIs) in live cells by means of a ribozyme complementation assay, but we found that the strategy was limited by the interaction affinity constraints and by low ribozyme activity in cells. We then sought to still detect interactions among endogenous proteins but in fixed cells. We devised an improved immunofluorescence (IF) technique, in which the antibodies are conjugated to an enzyme-substrate pair. We chose E. coli biotin ligase (BirA), which catalyzes the covalent ligation of biotin to a 15amino acid recognition sequence (AP). Only upon PPI would BirA be in close enough proximity to biotinylate the AP. Although the use of proximity biotinylation within the IF scheme proved challenging because of the geometric rigidity of the antibody conjugates, we later successfully applied the concept to the study of recombinant proteins in live cells, where BirA and AP were each genetically fused to the proteins of interest. We demonstrated that this method offers a combination of high spatial and temporal resolution with a low rate of false positives. We engineered the BirA/AP affinity to reduce background and eliminate false positives, while still allowing robust detection of relatively transient PPIs (half-life > 1 minute). We demonstrated that the methodology exhibits high specificity for the detection of PPIs in living mammalian cells, with a fold induction in the detected signal upon PPI of - 5-25. Using FRB-FKBP12 system as a model, the BirA/AP(-3) pair was also able to quantitatively predict interaction KIds. (cont.) Importantly, we showed that proximity biotinylation can detect the subcellular localization of the PPI under study. We also developed a new method for site-specific labeling of proteins in live cells. Through rational design, we re-directed E. coli lipoic acid ligase (LplA) to specifically ligate an unnatural alkyl azide substrate to an engineered 22-amino acid LplA acceptor peptide (LAP) tag. The alkyl azide can then be selectively derivatized with a cyclooctyne conjugated to any probe of interest. We first demonstrated that LplA can be used to label LAP-tagged proteins with Cy3, AlexaFluor568, and biotin at the surface of living mammalian cells, and we then applied the methodology to one- and two-color cellsurface receptor labeling. Finally, we also showed that LplA can site-specifically label intracellular proteins, although the signal/background ratio still needs to be improved. by Marta Fernández Suárez. Ph.D. 2009-03-16T19:28:05Z 2009-03-16T19:28:05Z 2008 2008 Thesis http://hdl.handle.net/1721.1/44678 260339666 eng 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 297 p. application/pdf Massachusetts Institute of Technology |
| spellingShingle | Chemistry. Fernández Suárez, Marta New reporters of protein trafficking and protein-protein interactions in live cells |
| title | New reporters of protein trafficking and protein-protein interactions in live cells |
| title_full | New reporters of protein trafficking and protein-protein interactions in live cells |
| title_fullStr | New reporters of protein trafficking and protein-protein interactions in live cells |
| title_full_unstemmed | New reporters of protein trafficking and protein-protein interactions in live cells |
| title_short | New reporters of protein trafficking and protein-protein interactions in live cells |
| title_sort | new reporters of protein trafficking and protein protein interactions in live cells |
| topic | Chemistry. |
| url | http://hdl.handle.net/1721.1/44678 |
| work_keys_str_mv | AT fernandezsuarezmarta newreportersofproteintraffickingandproteinproteininteractionsinlivecells |