Single-Molecule Conformational Motion and Atpase Activity of a AAA+ Proteolytic Machine

ClpXP is a hexameric AAA+ protease that binds, denatures and degrades protein substrates tagged for removal from the cell. Despite the many studies done on the ClpX unfoldase, it is still unknown how this motor couples ATP hydrolysis to mechanical movements to unravel a native protein and spool the polypeptide track into the ClpP degradation chamber. We used single-molecule fluorescence spectroscopy to investigate the conformational motions and ATPase activity within a single ClpX subunit. By using short-range fluorescence quenchers, we were able to resolve angstrom-level distance changes between domains and witness binding events of ATP analogs. This mechanistic study of ClpX helps to broaden our understanding of the large family of AAA+ motors and the novel techniques used here will advance the capabilities of single-molecule fluorescence measurements.