2026-09-19T15:02:22Zhttps://dspace.mit.edu/server/oai/requestoai:dspace.mit.edu:1721.1/460472022-01-13T07:54:21Zcom_1721.1_7582com_1721.1_7581col_1721.1_131022
Stuart Licht.
Jennings, Laura Danielle
Massachusetts Institute of Technology. Dept. of Chemistry.
Massachusetts Institute of Technology. Department of Chemistry
2009-06-30T17:08:36Z
2009-06-30T17:08:36Z
2008
2008
http://hdl.handle.net/1721.1/46047
370436103
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.
Vita.
Includes bibliographical references.
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.
(cont.)Both distributions contain peaks at integral multiples of 7-8 amino acids, consistent with a mechanism in which ClpP controls product sizes by alternating between translocation in steps of 7-8 amino acids and proteolysis. Collectively, the results presented here indicate that 1) conformational changes in both ClpA and ClpP are necessary for efficient translocation and proteolysis, 2) active site reactivity is linked to conformational changes in the pore region of ClpP, and 3) product sizes are largely controlled by ClpP.
by Laura Danielle Jennings.
Ph.D.
182 p.
eng
Massachusetts Institute of Technology
M.I.T. theses are protected by
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http://dspace.mit.edu/handle/1721.1/7582
Chemistry.
Translocation and proteolysis by the energy-dependent protease ClpAP : coordination of conformational changes and active site chemistry
Thesis
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<Abstract>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.</Abstract>
<Abstract>(cont.)Both distributions contain peaks at integral multiples of 7-8 amino acids, consistent with a mechanism in which ClpP controls product sizes by alternating between translocation in steps of 7-8 amino acids and proteolysis. Collectively, the results presented here indicate that 1) conformational changes in both ClpA and ClpP are necessary for efficient translocation and proteolysis, 2) active site reactivity is linked to conformational changes in the pore region of ClpP, and 3) product sizes are largely controlled by ClpP.</Abstract>
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