Mechanistic studies of the AAA+ molecular motor ClpXP
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2009.
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| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2009
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| Online Access: | http://hdl.handle.net/1721.1/49741 |
| _version_ | 1826213414122291200 |
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| author | Barkow, Sarah Rebecca |
| author2 | Robert T. Sauer. |
| author_facet | Robert T. Sauer. Barkow, Sarah Rebecca |
| author_sort | Barkow, Sarah Rebecca |
| collection | MIT |
| description | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2009. |
| first_indexed | 2024-09-23T15:48:42Z |
| format | Thesis |
| id | mit-1721.1/49741 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T15:48:42Z |
| publishDate | 2009 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/497412019-04-11T03:21:22Z Mechanistic studies of the AAA+ molecular motor ClpXP Barkow, Sarah Rebecca Robert T. Sauer. 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, 2009. Vita. Includes bibliographical references . ClpX is an archetypical representative of the AAA+ superfamily of enzymes that serves as the regulatory domain and motor for the ClpXP protease system. ClpX binds protein substrates via an amino acid sequence known as a tag, denatures them, and translocates them into the associated peptidase, ClpP. ClpX utilizes the energy from ATP hydrolysis to pull on bound substrates, destabilizing folded substrates and denaturing them, before they are translocated through the pore of ClpX into ClpP. As a representative of the AAA+ superfamily, mechanistic understanding of the ClpXP protease cycle may elucidate the mechanisms of related enzymes. I used synthetic peptide substrates to probe what features of a polypeptide chain are recognized during translocation. Surprisingly, side-chain properties including size and charge, and the spacing between peptide bonds had relatively small effects on the rates of translocation by ClpXP. Pulling on tracts of glycine, lysine, or proline also allowed efficient ClpXP degradation of the stable protein GFP, for which unfolding is rate limiting. These results suggest that minimal chemical or structural features may be sufficient for translocation and protein unfolding by ClpX and lead to a new model for translocation based on multiple van der Waal's interactions. ClpX interacts with its substrates via highly conserved pore loops including the GYVG pore-i loop. I identify that the hydroxyl group of the tyrosine, Y153, is directly involved in translocation. (cont.) In addition, I show Y153 is involved in binding substrates and is essential for coupling ATPase activity and translocation. I investigate substrate tag recognition by ClpX and the role of Y 153 in substrate binding via a library of synthetic peptides. In the ClpX recognition tag, ssrA (AANDENYALAA), the last residue plays the largest role in recognition and the penultimate residue plays a smaller role. The incorporation of charges significantly disrupts binding and chirality is essential. ClpX is a versatile enzyme, able to translocate substrates via the highly permissive and novel mechanism of using van der Waal's interactions. Selectivity and control are exerted on the level of substrate specificity, where ClpX displays high specificity for certain substrate tag sequences that are not tolerant of modification. by Sarah Rebecca Barkow. Ph.D. 2009-11-06T16:28:09Z 2009-11-06T16:28:09Z 2009 2009 Thesis http://hdl.handle.net/1721.1/49741 454516993 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 | Chemistry. Barkow, Sarah Rebecca Mechanistic studies of the AAA+ molecular motor ClpXP |
| title | Mechanistic studies of the AAA+ molecular motor ClpXP |
| title_full | Mechanistic studies of the AAA+ molecular motor ClpXP |
| title_fullStr | Mechanistic studies of the AAA+ molecular motor ClpXP |
| title_full_unstemmed | Mechanistic studies of the AAA+ molecular motor ClpXP |
| title_short | Mechanistic studies of the AAA+ molecular motor ClpXP |
| title_sort | mechanistic studies of the aaa molecular motor clpxp |
| topic | Chemistry. |
| url | http://hdl.handle.net/1721.1/49741 |
| work_keys_str_mv | AT barkowsarahrebecca mechanisticstudiesoftheaaamolecularmotorclpxp |