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.

Project Report

Protein integrity inside cells is maintained by a series of tightly regulated and controlled processes that can unfold proteins, dismantle multimers, and solubilize aggregates. An AAA+ protease known as ClpXP is a model system for understanding AAA+ motors whose machinery is found in all cells performing a wide range of tasks. This molecular machine recognizes, denatures and degrades proteins that have been targeted for decomposition in bacterial cells. ClpXP is composed of the ring shaped ATPase ClpX, which mechanically unfolds and translocates polypeptides, delivering them into the peptidase ClpP, which degrades unfolded substrates.Through support from my NSF-sponsored CAREER award, my lab and our collaborators investigated the ClpXP molecular motor at the single molecule level leading to assays where one directly observes machinery tasks. We pioneered the first single molecule fluorescence assay and single molecule mechanical assay to directly monitor functioning ClpXP machinery. This work begins to address fundamental questions regarding how molecular machines harness ATP to dismantle protein structures. The work also characterized motility on a polypeptide based track. The project strengthened a link between a team of researchers bringing together expertise in single molecule biophysics with collaborators who are expert in protein chemistry and molecular biology. Although the major impact was the direct work on ClpXP machinery, the award also enabled studies of amyloid fibers, actin machinery, T-cell triggering, peptide aptamers, and methods development including combined trapping and fluorescence and stochastic optical active microrheology. Specifically related to education, the project resulted in the development of an undergraduate optical trap used in core teaching labs. The instrument design has been adopted in many labs across the country. We and others working with such teaching-dedicated instruments also share ideas about lab modules that have been implemented successfully including motility assays, DNA tethering, calibrations etc. mostly focused on introducing concepts of single molecule biophysics to undergraduates. NSF support to my lab has been used to directly train graduate students from underrepresented groups at an advanced level. My lab has trained a number of students, postdocs and affiliates who have gone on to launch independent academic careers, careers in industry and related disciplines.

Agency
National Science Foundation (NSF)
Institute
Division of Molecular and Cellular Biosciences (MCB)
Application #
1118490
Program Officer
Michele McGuirl
Project Start
Project End
Budget Start
2010-09-01
Budget End
2013-08-31
Support Year
Fiscal Year
2011
Total Cost
$241,281
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
DUNS #
City
Nashville
State
TN
Country
United States
Zip Code
37240