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Mechanically Watching the ClpXP Proteolytic Machinery.
Juan Carlos Cordova1, Adrian O Olivares2, Matthew J Lang3
1Department of Chemical and Biomolecular Engineering, Vanderbilt University, 308-A Olin Hall, VU Mailbox: PMB 351604, Nashville, TN, 37235, USA.
Methods in Molecular Biology (Clifton, N.J.)
|November 16, 2016
Summary
The ClpXP motility assay uses dual optical traps to track protein unfolding and degradation. This method reveals detailed steps in energy-dependent protein breakdown, applicable to various protease systems.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Energy-dependent protein degradation is crucial for cellular regulation.
- The ClpXP protease system plays a key role in degrading misfolded or damaged proteins.
- Understanding the mechanical steps of protein degradation is essential for elucidating its function.
Purpose of the Study:
- To develop and validate a dual bead ClpXP motility assay for studying protein degradation.
- To characterize the mechanical subprocesses involved in folded protein processing by ClpXP.
- To establish a versatile platform for analyzing diverse protein substrates and protease machinery.
Main Methods:
- Utilizing a dual optical trap in a passive force-clamp configuration.
- Employing bead-to-bead displacement measurements to monitor protein unfolding and translocation.
- Designing engineered protein substrates and modifying the ClpXP protease machinery.
Main Results:
- The assay successfully resolved discrete nanometer-scale displacements corresponding to unfolding and translocation substeps.
- Observed steps, dwells, and pauses provide insights into the kinetics and mechanics of degradation.
- Demonstrated the assay's adaptability as a chassis for various substrates and related machinery.
Conclusions:
- The dual bead ClpXP motility assay provides a high-resolution method for dissecting energy-dependent protein degradation.
- This technique offers a powerful tool for quantitative analysis of protein processing by proteases.
- The adaptable nature of the assay facilitates its application to a broad spectrum of protein degradation systems.
