Translocation and proteolysis by the energy-dependent protease ClpAP : coordination of conformational changes and active site chemistry

Energy-dependent proteases, such as the E. coli protease ClpAP, degrade misfolded and short-lived regulatory proteins inside the cell. In this class of proteins, an ATPase component (e.g., ClpA) binds, unfolds, and translocates protein substrates into a protease component (e.g., ClpP) where degradation occurs. This thesis addresses the communication between ClpA and ClpP required to efficiently translocate and degrade protein substrates. Synchrotron hydroxyl radical footprinting is used to show that the ClpA D2 loop, located in the pore of ClpA, is in an "up" conformation when ATP is bound and assumes a "down" conformation when ADP is bound. These results provide the first direct experimental support for a nucleotide-dependent D2 loop conformational change previously proposed to mediate substrate translocation. Synchrotron footprinting also shows that the ClpP N-terminal loops undergo a conformational change, upon the binding of ClpA, from a closed, pore-blocking position, to an open, pore-free position. Complementary kinetic studies show that deletion of the ClpP N-terminus (ClpPAN) accelerates the degradation rate of large peptide substrates 5-15 fold, indicating that opening of the pore is functionally important. Furthermore, unlike ClpAP and wild-type ClpP, ClpPAN shows a distinct slow phase of product formation that is eliminated with the addition of hydroxylamine, suggesting that deletion of the N-terminal loops stabilizes the acyl-enzyme intermediate. Additionally, size-exclusion chromatography and kinetic studies are used to show that ClpP alone can processively degrade a full-length protein substrate in the absence of ClpA, albeit at a 2000-fold slower rate, and furthermore that the size distribution of ClpP-generated peptide products is strikingly similar to the size distribution of ClpAP-generated peptide products.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2008

Author dc:creator

• Jennings, Laura Danielle

Advisor dc:contributor.advisor

• Stuart Licht.

• 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/1721.1/46047

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/46047