Specificity and regulation of substrate degradation for a AAA+ protease
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007.
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| Other Authors: | |
| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2007
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| Online Access: | http://hdl.handle.net/1721.1/38998 |
| _version_ | 1826192145181048832 |
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| author | Farrell, Christopher Mark |
| author2 | Robert T. Sauer. |
| author_facet | Robert T. Sauer. Farrell, Christopher Mark |
| author_sort | Farrell, Christopher Mark |
| collection | MIT |
| description | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007. |
| first_indexed | 2024-09-23T09:06:38Z |
| format | Thesis |
| id | mit-1721.1/38998 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T09:06:38Z |
| publishDate | 2007 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/389982019-04-10T20:50:03Z Specificity and regulation of substrate degradation for a AAA+ protease Farrell, Christopher Mark Robert T. Sauer. 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, 2007. Includes bibliographical references. Energy dependent proteolysis is a critical method of cellular regulation for all forms of life. The AAA+ proteases ClpXP and ClpAP in E. coli function in this capacity by facilitating the denaturation and degradation of target substrates. These proteolytic enzymes degrade hundreds of different proteins. Determining how the activities of these proteases are regulated in the cell as well as learning how these enzymes bind and engage substrates are important goals. In order to better understand how the degradation of ClpXP and ClpAP is regulated, I studied their contributions to ssrA-tagged protein degradation in the cell. Using GFP-ssrA expressed from the chromosome as a degradation reporter, the effects of altered concentrations of different protease components or adaptor proteins were explored. I found that both ClpXP and ClpAP could degrade GFP-ssrA in the cell and that increased levels of ClpAP in stationary phase resulted in increased degradation of ssrA-tagged substrates. I also demonstrated that wild-type levels of the adaptor proteins SspB and ClpS do not fully inhibit ClpAP degradation of GFP-ssrA. To better understand how the ClpXP enzyme binds substrates, I took a mutagenic approach. (cont.) The "RKH" loops surround the entrance to the central pore of the ClpX hexamer and are highly conserved in the ClpX subfamily of AAA+ ATPases. I discovered that a mutation within the RKH loop of ClpX changes substrate specificity by 300-fold, resulting in decreased degradation of ssrA-tagged substrates but improved degradation of proteins with other classes of degradation signals. My results show that the RKH loops recognize the C-terminal carboxylate of the ssrA tag and suggest that ClpX specificity represents an evolutionary compromise that has optimized degradation of multiple types of substrates rather than any single class. by Christopher Mark Farrell. Ph.D. 2007-09-28T13:29:55Z 2007-09-28T13:29:55Z 2007 2007 Thesis http://hdl.handle.net/1721.1/38998 166527112 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 115 leaves application/pdf Massachusetts Institute of Technology |
| spellingShingle | Biology. Farrell, Christopher Mark Specificity and regulation of substrate degradation for a AAA+ protease |
| title | Specificity and regulation of substrate degradation for a AAA+ protease |
| title_full | Specificity and regulation of substrate degradation for a AAA+ protease |
| title_fullStr | Specificity and regulation of substrate degradation for a AAA+ protease |
| title_full_unstemmed | Specificity and regulation of substrate degradation for a AAA+ protease |
| title_short | Specificity and regulation of substrate degradation for a AAA+ protease |
| title_sort | specificity and regulation of substrate degradation for a aaa protease |
| topic | Biology. |
| url | http://hdl.handle.net/1721.1/38998 |
| work_keys_str_mv | AT farrellchristophermark specificityandregulationofsubstratedegradationforaaaaprotease |