Skip to main content
Open Access Publications from the University of California

UC Berkeley

UC Berkeley Electronic Theses and Dissertations bannerUC Berkeley

Mechanism of Protein Unfolding and Polypeptide Translocation by the AAA+ Protease ClpXP


ATP-dependent proteases exist in all cells and are crucial regulators of the pro-

teome. These biological nanomachines contain a homohexameric molecular motor

which generates mechanical force to unfold proteins and translocate unstructured

polypeptides into a degradation chamber for proteolysis. Using E. coli ClpXP as

a model system for investigating the function of ATP-dependent proteases, I used

single-molecule optical tweezers to explore how individual subunits within the hex-

americ motor convert the chemical energy from ATP hydrolysis into the mechanical

work required to perform protein unfolding and polypeptide translocation.

These studies revealed key aspects of motor function that highlight how ATP-

dependent proteases are tailored to serve their specific cellular tasks. My analyses

indicated that ClpXP translocates in bursts resulting from highly coordinated con-

formational changes in two to four ATPase subunits and that these bursts of translo-

cation couple to the phosphate release step of the ATP hydrolysis cycle. I found

that firing of four ATPase subunits is required to successfully unfold GFP, but that

polypeptide translocation robustly occurs even when only two ATPase subunits fire.

Because this work pushed the envelope of single-molecule data analysis techniques,

I developed new algorithms to detect steps in passive-mode optical tweezers data,

where the noise statistics vary in time.

The results discussed in this dissertation improve our understanding of how ClpXP

mechanically operates. Strong structural similarities to other ATP-dependent pro-

teases, as well as other ring translocases, suggest that the operating principles I have

discerned and describe here may generalize to other biological molecular motors.

Main Content
For improved accessibility of PDF content, download the file to your device.
Current View