(158d) Single-Molecule Fluorescence Quenching Reveals Conformational Dynamics of the AAA+ Protease ClpXP

AAA+ proteases maintain protein quality control and help regulate cellular processes through targeted protein degradation. ClpXP from Escherichia coli is a model AAA+ protease that uses ATP-powered conformational motions to unfold, translocate, and degrade proteins labeled with specific tags. ClpX serves as a gatekeeper: unfolding and translocating proteins before threading the unraveled polypeptide into the lumen of ClpP for degradation. Despite numerous structural and mechanistic studies of ClpXP, the conformational states and motions in a working ClpXP motor have not yet been characterized. Here, we developed a novel, short-range single-molecule fluorescence quenching assay to monitor the functional motions of the ClpX machine bound to a colocalized ClpP molecule. The short-range sensitivity (R₀ ~ 2.6 nm) of our assay paired with distance calibration measurements using a DNA scaffold permit quantitative reporting of the conformational state and transitions in a single subunit of ClpX. Our results show that ClpXP cycles through dynamic and static conformational modes under varying nucleotide and substrate conditions. Additionally, we observed conformational switching in the absence of nucleotides suggesting that some structural changes are thermally driven. This method enables real-time observation of the structural dynamics of working AAA+ machines and provides context and mechanistic insight for recently solved cryogenic electron microscopy (cryoEM) structures of ClpXP^1–3. Our technique can be adapted to study the movements and interactions of a host of molecular motors and other biomolecules where short-range conformational changes occur.

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