(334s) Molecular Mechanisms of the AAA+ Protease Clpxp Characterized Using Single-Molecule Fluorescence Quenching

AAA+ proteases are enzymes responsible for removing unwanted proteins and activating cellular processes through the degradation of tagged proteins. Escherichia coli ClpXP is a model AAA+ protease that uses the energy of ATP binding and hydrolysis to fuel protein degradation. The hexameric ClpX ring first unfolds a specific protein before translocating the polypeptide into the ClpP degradation chamber. Various biochemical and structural studies have been used to characterize ClpXP, yet the conformational transitions and nucleotide transactions of ClpXP subunits are still unknown. Here, we engineered two novel, short-range single-molecule fluorescence quenching assays to monitor conformational switching and nucleotide-binding in a single subunit of ClpXP. We synthesized an ATP analog labeled with a dark quencher that we call ATPQ for our single-molecule nucleotide-binding assay. ATPQ supports ClpXP motor activity and permits the observation of binding events at concentrations up to 100 μM. Our conformational switching assay and DNA-based distance calibrations allow for quantitative measurements of conformational states and dynamics of individual ClpX subunits. Our results show that ClpXP subunits adopt both static and dynamic conformational states at varying nucleotide and substrate conditions. The single-molecule techniques we developed here facilitate real-time observation of molecular events in working AAA+ motors and can be applied to a wide range of molecular motors.

Research Interests

Biophysics, Biochemistry, Molecular Biology, Optical Trapping, Protein Engineering, Optics, Photonics, Fluorescence, Single-Molecule Biophysics, AAA+ Motors