Role of communication between subunits and enzymes in ClpXP-mediated substrate unfolding and degradation

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004.

Bibliographic Details
Main Author: Joshi, Shilpa Arun, 1975-
Other Authors: Robert T. Sauer.
Format: Thesis
Language:eng
Published: Massachusetts Institute of Technology 2005
Subjects:
Online Access:http://hdl.handle.net/1721.1/16608
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author Joshi, Shilpa Arun, 1975-
author2 Robert T. Sauer.
author_facet Robert T. Sauer.
Joshi, Shilpa Arun, 1975-
author_sort Joshi, Shilpa Arun, 1975-
collection MIT
description Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2004.
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spelling mit-1721.1/166082019-04-11T08:15:58Z Role of communication between subunits and enzymes in ClpXP-mediated substrate unfolding and degradation Mechanistic studies of Escherichia coli CLPXP's substrate unfolding and degradation cycles Joshi, Shilpa Arun, 1975- 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, 2004. Vita. Includes bibliographical references. This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. Chaperones and proteases play important roles in quality control by helping proteins fold, by dismantling hyper-stable complexes, and by degrading unwanted proteins. The highly conserved AAA+ Clp/Hsp100 proteins are ATPases which function as disassembly chaperones as well as essential components of energy-dependent proteases. For example, the ClpX ATPase disassembles macromolecular complexes and combines with the ClpP peptidase to form ClpXP, a molecular machine with structural and functional similarity to the eukaryotic 26S proteasome. ClpXP consists of hexameric ClpX rings stacked coaxially against the double-ring ClpP₁₄ peptidase. ClpXP's peptidase active sites reside in a sequestered chamber accessible through a narrow channel which excludes native, folded substrates. ClpX binds specific substrates, unfolds them in a reaction requiring ATP hydrolysis, and then translocates them into ClpP for degradation. When ClpP is absent, ClpX unfolds and releases specific substrates, an activity that can disassemble otherwise stable complexes. Although ClpX-mediated substrate unfolding and ClpXP-mediated degradation have been studied extensively, the role of communication between subunits within a ClpX hexamer or between enzymes in ClpXP has not been addressed. I initially screened numerous ClpX point mutants for their ability to support host-cell lysis by bacteriophage Mu, and then purified several mutants for functional characterization in vitro. Three of these mutants contained substitutions at intersubunit interfaces; these variants bound substrate well, but displayed unusual changes in ATP hydrolysis in response to substrate binding and had low protein unfolding activity. (cont.) These results suggest that communication across these subunit-subunit interfaces coordinates and regulates nucleotide hydrolysis in response to substrate binding and is critical for efficient substrate unfolding. I also discovered that ClpX and ClpP communicate with each other during substrate processing and degradation. ClpX binds ClpP more strongly during substrate unfolding than during translocation, by a mechanism that is linked to the ATP hydrolysis rate. Interaction between ClpX and DFP-modified ClpP, mimicking a peptide-cleavage intermediate, is strengthened significantly suggesting that ClpP also communicates its functional state to ClpX. Overall, this work suggests that communication within the ClpX hexamer and between enzymes in ClpXP modulates and coordinates substrate processing. by Shilpa Arun Joshi. Ph.D. 2005-05-17T14:39:19Z 2005-05-17T14:39:19Z 2004 2004 Thesis http://hdl.handle.net/1721.1/16608 55634747 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 137 leaves 5240713 bytes 8858604 bytes application/pdf application/pdf application/pdf Massachusetts Institute of Technology
spellingShingle Biology.
Joshi, Shilpa Arun, 1975-
Role of communication between subunits and enzymes in ClpXP-mediated substrate unfolding and degradation
title Role of communication between subunits and enzymes in ClpXP-mediated substrate unfolding and degradation
title_full Role of communication between subunits and enzymes in ClpXP-mediated substrate unfolding and degradation
title_fullStr Role of communication between subunits and enzymes in ClpXP-mediated substrate unfolding and degradation
title_full_unstemmed Role of communication between subunits and enzymes in ClpXP-mediated substrate unfolding and degradation
title_short Role of communication between subunits and enzymes in ClpXP-mediated substrate unfolding and degradation
title_sort role of communication between subunits and enzymes in clpxp mediated substrate unfolding and degradation
topic Biology.
url http://hdl.handle.net/1721.1/16608
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