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Using Microtiter Dish Radiolabeling for Multiple In Vivo Measurements Of Escherichia coli (p)ppGpp Followed by Thin Layer Chromatography
Published on: June 4, 2019
E. coli ClpA catalyzed polypeptide translocation is allosterically controlled by the protease ClpP
Justin M Miller1, Jiabei Lin, Tao Li
1Department of Chemistry, The University of Alabama at Birmingham, 1530 Third Avenue South, Birmingham, AL 35294-1240, USA.
Journal of Molecular Biology
|May 4, 2013
Summary
The ClpP protease allosterically affects ClpA
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Degradation
Background:
- Escherichia coli possesses five ATP-dependent proteases crucial for protein quality control.
- ClpA unfolds and translocates substrates into the ClpP protease core for degradation.
- The allosteric impact of ClpP on ClpA's translocation mechanism was previously uncharacterized.
Purpose of the Study:
- To investigate the allosteric effect of ClpP on ClpA-mediated polypeptide translocation.
- To elucidate the mechanistic changes in ClpA activity upon association with ClpP.
Main Methods:
- Utilized single-turnover fluorescence stopped-flow methods.
- Quantified translocation rates and kinetic step sizes.
- Analyzed cooperativity between ATP binding sites in ClpA.
Main Results:
- ClpA translocates polypeptides at ~35 amino acids per second when associated with ClpP.
- Translocation involves traversing ~5 amino acids between rate-limiting steps with reduced ATP site cooperativity.
- In contrast, ClpA alone translocates at ~20 amino acids per second with higher ATP site cooperativity.
Conclusions:
- ClpP binding allosterically modulates ClpA's polypeptide translocation.
- ClpP reduces cooperativity between ClpA's ATP binding sites, enhancing translocation efficiency.
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