Determining protein interaction specificity of native and designed bZIP family transcription factors
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.
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| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2012
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| Online Access: | http://hdl.handle.net/1721.1/70392 |
| _version_ | 1826188074007134208 |
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| author | Reinke, Aaron W |
| author2 | Amy Keating. |
| author_facet | Amy Keating. Reinke, Aaron W |
| author_sort | Reinke, Aaron W |
| collection | MIT |
| description | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012. |
| first_indexed | 2024-09-23T07:54:11Z |
| format | Thesis |
| id | mit-1721.1/70392 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T07:54:11Z |
| publishDate | 2012 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/703922019-04-09T15:25:05Z Determining protein interaction specificity of native and designed bZIP family transcription factors Reinke, Aaron W Amy Keating. Massachusetts Institute of Technology. Dept. of Biology. Massachusetts Institute of Technology. Dept. of Biology. Biology. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012. Page 428 blank. Cataloged from PDF version of thesis. Includes bibliographical references. Protein-protein interactions are important for almost all cellular functions. Knowing which proteins interact with one another is important for understanding protein function as well as for being able to disrupt their interactions. The basic leucine-zipper transcription factors (bZIPs) are a class of eukaryotic transcription factors that form either homodimers or heterodimers that bind to DNA in a site-specific manner. bZIPs are similar in sequence and structure, yet bZIP protein-protein interactions are specific, and this specificity is important for determining which DNA sites are bound. bZIP proteins have a simple structure that makes them experimentally tractable and well suited for developing models of interaction specificity. While current models perform well at being able to distinguish interactions from non-interactions, they are not fully accurate or able to predict interaction affinity. Our current understanding of protein interaction specificity is limited by the small number of large, high-quality interaction data sets that can be analyzed. For my thesis work I took a biophysical approach to experimentally measure the interactions of many native and designed bZIP and bZIP-like proteins in a high-throughput manner. The first method I used involved protein arrays containing small spots of bZIP-derived peptides immobilized on glass slides, which were probed with fluorescently labeled candidate protein partners. To improve upon this technique, I developed a solution-based FRET assay. In this experiment, two different dye-labeled versions of each protein are purified and mixed together at multiple concentrations to generate binding curves that quantify the affinity of each pair-wise interaction. Using the array assay, I identified novel interactions between human proteins and virally encoded bZIPs, characterized peptides designed to bind specifically to native bZIPs, and measured the interactions of a large set of synthetic bZIP-like coiled coils. Using the solution-based FRET assay, I quantified the bZIP interaction networks of five metazoan species and observed conservation as well as rewiring of interactions throughout evolution. Together, these studies have identified new interactions, created peptide reagents, identified sequence determinants of interaction specificity, and generated large amounts of interaction data that will help in the further understanding of bZIP protein interaction specificity. by Aaron W. Reinke. Ph.D. 2012-04-26T18:49:17Z 2012-04-26T18:49:17Z 2012 2012 Thesis http://hdl.handle.net/1721.1/70392 783810564 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 428 p. application/pdf Massachusetts Institute of Technology |
| spellingShingle | Biology. Reinke, Aaron W Determining protein interaction specificity of native and designed bZIP family transcription factors |
| title | Determining protein interaction specificity of native and designed bZIP family transcription factors |
| title_full | Determining protein interaction specificity of native and designed bZIP family transcription factors |
| title_fullStr | Determining protein interaction specificity of native and designed bZIP family transcription factors |
| title_full_unstemmed | Determining protein interaction specificity of native and designed bZIP family transcription factors |
| title_short | Determining protein interaction specificity of native and designed bZIP family transcription factors |
| title_sort | determining protein interaction specificity of native and designed bzip family transcription factors |
| topic | Biology. |
| url | http://hdl.handle.net/1721.1/70392 |
| work_keys_str_mv | AT reinkeaaronw determiningproteininteractionspecificityofnativeanddesignedbzipfamilytranscriptionfactors |