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 motorwhich generates mechanical force to unfold proteins and translocate unstructuredpolypeptides into a degradation chamber for proteolysis. Using E. coli ClpXP asa model system for investigating the function of ATP-dependent proteases, I usedsingle-molecule optical tweezers to explore how individual subunits within the hex-americ motor convert the chemical energy from ATP hydrolysis into the mechanicalwork 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 analysesindicated 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 foundthat firing of four ATPase subunits is required to successfully unfold GFP, but thatpolypeptide 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 ClpXPmechanically operates. Strong structural similarities to other ATP-dependent pro-teases, as well as other ring translocases, suggest that the operating principles I havediscerned and describe here may generalize to other biological molecular motors