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Related Experiment Video

Updated: Jul 17, 2026

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Published on: November 23, 2016

Altered specificity of a AAA+ protease.

Christopher M Farrell¹, Tania A Baker, Robert T Sauer

¹Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

|January 16, 2007

Summary

Researchers identified a ClpX mutant altering substrate specificity. This mutation in RKH loops enhances degradation of some proteins while decreasing others, suggesting an evolutionary compromise in ClpXP protease function.

Area of Science:

Biochemistry
Molecular Biology
Proteolysis

Background:

ClpXP is an ATP-dependent protease crucial for intracellular protein degradation.
Substrate recognition by ClpX involves binding to peptide tags, typically at protein termini.

Purpose of the Study:

To identify mutations affecting ClpX substrate specificity.
To investigate the role of RKH loops in ClpX substrate recognition.

Main Methods:

Site-directed mutagenesis to create ClpX mutants.
In vitro assays to measure protein degradation rates.

Main Results:

A ClpX mutant showed a 300-fold change in substrate specificity.
Degradation of ssrA-tagged substrates decreased, while other tagged substrates were degraded more efficiently.

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The mutation was mapped to the conserved RKH loops at the ClpX pore entrance.

Conclusions:

RKH loops play a critical role in ClpX substrate recognition.
ClpX specificity is likely an evolutionary compromise optimizing degradation of diverse substrates.