Single-Molecule Protein Unfolding and Translocation by an ATP-Fueled Proteolytic Machine

SummaryAll cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5–8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (314 K)Download as PowerPoint slideHighlights► Single-molecule nanometry reveals the mechanics of ClpXP-mediated protein unfolding ► ClpXP pulls polypeptides apart, performing more than 5 kT of work per power stroke ► Protein unfolding is fast and highly cooperative, but can require many ATPase cycles ► The smallest translocation steps for ClpX and ClpXP range from 5–8 amino acids