Mechanistic studies of the AAA+ molecular motor ClpXP

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.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2009

Author dc:creator

• Barkow, Sarah Rebecca

Advisor dc:contributor.advisor

• Robert T. Sauer.

• Chemistry.

Statement dc:rights

• 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.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/49741

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/49741