CAREER: ClpXP Machinery Studied at the Single-Molecule Level

Massachusetts Institute Of Technology, Cambridge MA

Investigators

Abstract

Protein integrity inside cells is maintained by a series of tightly regulated processes that denature proteins, dismantle multimers, and solubilize aggregates. A key component of this regulation in bacterial systems is the ClpXP protease, which is composed of the ClpX ATPase and the ClpP peptidase, both members of the Clp/Hsp100 chaperone family. ClpXP regulation is a highly coordinated process by which ClpX recognizes a substrate that has been labeled with a specific amino acid sequence, mechanically unfolds it into a linear polypeptide, and ultimately translocates it into ClpP for irreversible proteolysis. While the sequence of this process was recently established, the molecular details of the mechanical cycle are poorly understood. This project aims to elucidate the mechanochemical mechanism by which ClpXP uses ATP to mechanically unfold and translocate substrates. To this end, single molecule fluorescence and optical tweezers-based assays will be developed and employed for detailed measurements of ClpXP motility. Such quantitative studies of this machinery will explore the strength of the enzyme-substrate interaction; the forces and speeds at which ClpX unfolds and translocates a variety of different substrates; the efficiency by which ClpX converts ATP into mechanical energy; and the intermolecular coordination of ClpX with ClpP. These results will provide the first direct observation of protein-mediated mechanical unfolding and will yield insight into other cellular processes and mechanisms associated with proteolytic degradation. This project will explore a new class of biological machinery distinguished by high force landscapes and biomechanical denaturing and digestion of cellular proteins. In addition, the novel motor motility track uniquely runs on a polypeptide backbone, in contrast to other well studied motors that move along filaments of actin and microtubules or polymers of DNA and RNA. Broader Impacts The scientific impact of this project will be complemented by an educational component focusing on the development of hands-on biophysics laboratory training for undergraduates. Laboratory modules will be developed and implemented to leverage the undergraduate optical traps developed by the PI with the classical kinesin motility assay and a new ClpXP motility assay. These educational initiatives will provide students with direct training experience in biological motor motility assays and biophysics instrumentation and will be adapted for summer laboratory courses and through the development of a web-based wiki portal. Combined with these educational and outreach activities, this work will advance the study of mechanical forces in biology and contribute to the understanding of Nature's molecular machinery.