Nucleotide binding and conformational switching in the hexameric ring of a AAA+ machine
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, February 2015.
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | eng |
| Published: |
Massachusetts Institute of Technology
2015
|
| Subjects: | |
| Online Access: | http://hdl.handle.net/1721.1/97273 |
| _version_ | 1826200158395695104 |
|---|---|
| author | Stinson, Benjamin M. (Benjamin Michael) |
| author2 | Robert T. Sauer and Tania A. Baker. |
| author_facet | Robert T. Sauer and Tania A. Baker. Stinson, Benjamin M. (Benjamin Michael) |
| author_sort | Stinson, Benjamin M. (Benjamin Michael) |
| collection | MIT |
| description | Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, February 2015. |
| first_indexed | 2024-09-23T11:32:04Z |
| format | Thesis |
| id | mit-1721.1/97273 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T11:32:04Z |
| publishDate | 2015 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/972732019-04-11T06:29:41Z Nucleotide binding and conformational switching in the hexameric ring of a AAA+ machine Stinson, Benjamin M. (Benjamin Michael) Robert T. Sauer and Tania A. Baker. Massachusetts Institute of Technology. Department of Biology. Massachusetts Institute of Technology. Department of Biology. Biology. Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, February 2015. This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. Cataloged from student-submitted PDF version of thesis. Includes bibliographical references. ATP-powered proteases enforce protein quality-control and regulation in all domains of life. ClpX, a AAA+ ring homohexamer, uses the energy of ATP binding and hydrolysis to power conformational changes that unfold and translocate target proteins into the ClpP peptidase for degradation. X-ray crystal structures show that some ClpX subunits occupy nucleotide-loadable conformations and others occupy unloadable conformations. Furthermore, biochemical evidence suggests that multiple classes of nucleotide-loadable subunits exist. How asymmetry among subunits is coordinated to achieve mechanical function has remained unclear. Using a combination of mutagenesis, disulfide crosslinking, and fluorescence methods to assay the conformations and nucleotide-binding properties of individual subunits, we demonstrate dynamic interconversion of loadable and unloadable subunits. Such interconversion is required to couple ATP hydrolysis by ClpX to mechanical work, plays a role in substrate binding and ClpP interaction, and is not strictly coupled to the ATP hydrolysis cycle. ATP binding to different classes of subunits drives allosteric changes in ring conformation to allow hydrolysis and coupled machine function, and we present a subunit-specific single molecule nucleotide occupancy assay to elucidate details of this process. by Benjamin M. Stinson. Ph. D. 2015-06-10T18:42:07Z 2015-06-10T18:42:07Z 2014 2015 Thesis http://hdl.handle.net/1721.1/97273 910720311 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 140 pages application/pdf Massachusetts Institute of Technology |
| spellingShingle | Biology. Stinson, Benjamin M. (Benjamin Michael) Nucleotide binding and conformational switching in the hexameric ring of a AAA+ machine |
| title | Nucleotide binding and conformational switching in the hexameric ring of a AAA+ machine |
| title_full | Nucleotide binding and conformational switching in the hexameric ring of a AAA+ machine |
| title_fullStr | Nucleotide binding and conformational switching in the hexameric ring of a AAA+ machine |
| title_full_unstemmed | Nucleotide binding and conformational switching in the hexameric ring of a AAA+ machine |
| title_short | Nucleotide binding and conformational switching in the hexameric ring of a AAA+ machine |
| title_sort | nucleotide binding and conformational switching in the hexameric ring of a aaa machine |
| topic | Biology. |
| url | http://hdl.handle.net/1721.1/97273 |
| work_keys_str_mv | AT stinsonbenjaminmbenjaminmichael nucleotidebindingandconformationalswitchinginthehexamericringofaaaamachine |