ClpXP belongs to a family of motors known as AAA+. These predominantly ring shaped mechanoenzymes hydrolyze ATP to perform a diverse set of tasks throughout the cell such as transport, remodeling, degradation, and control over oscillation, gene expression and even inheritance. AAA+ motors are found in all kingdoms of life and count their numbers among 30,000 whose genes have been identified to date. Cells maintain the quality of proteins within their walls by a series of tightly regulated and controlled processes. They also have the ability to control what proteins are present by dismantling and otherwise solubilizing unwanted species. One system that accomplishes this task is the protein degradation machinery, which is a series of linked processes that recognize, denature and degrade proteins that have been targeted for decomposition. In bacterial cells this task is performed by ClpXP, a model molecular motor complex. Organized as a stacked series of ring-like structures, ClpXP processes proteins along the central axis of the rings. Subsystems of recognition, unfolding, translocation and degradation are coordinated at different points along the rings. Like a conventional motor, ClpXP utilizes its fuel molecule, ATP, to physically apply forces to target proteins, ultimately resulting in their dismantling. ClpXP then translocates these proteins into a chamber where degradation occurs. This study is aimed at elucidating how ClpXP harnesses ATP to perform complex tasks such as protein denaturation and how it coordinates the process of recognition, translocation, denaturation and degradation. The approach taken in this project would isolate single ClpXP motors where the progress of unfolding and translocation are separately and directly observed with advanced custom-built microscopes for single molecule detection. With the use of this technique, minute forces, piconewtons, and tiny displacements, in the realm of nanometers, are measured to quantify motor output. A series of model proteins with varying natural integrity and stabilities serve as a systematic way to measure motor output. Situations where external forces are applied with and against the molecular motor output will also be researched. Subdomain motions underlying mechanical action of the motor will be directly observed. This project brings together expertise in single-molecule biophysics with collaborators who are experts in protein chemistry and molecular biology. The project will ultimately lead to the development of novel fluorescence and mechanical assays that are beneficial for studying biological machinery in general.
Broader Impacts:
The educational objectives of the project are tied to hands-on lab-based instruction. The first called "go-forth-and-measure" is a program designed to reach undergraduate students in a way that excites them to plan and implement measurements of their own design. Students are provided with a selection of sensors and measurement methods to design and execute an experiment of their choosing, one that captivates their imagination. The second program is "building-with-biology" where students create in the prototyping space of physical biological parts, for example, through combining parts from biological motors and cell machinery. Students will share methods and strategies for prototyping with biological components so they can leverage one another's work. Both initiatives will be promoted at national meetings that bring together physics research and education teachers tied to education at the interface of physics and biology.
