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Dissecting the molecular mechanisms of the ClpXP protease, one molecule at a time

  • Author(s): Sen, Maya
  • Advisor(s): Bustamante, Carlos
  • et al.
Abstract

The cell is a fundamental unit of life, and in order for survival, maintaining a stable intracellular enviroment is crucial. ATP-dependent protease complexes regulate protein quality and abundance to ensure homeostasis in the cell. They use chemical energy to power processes necessary for regulating the intracellular environment including protein unfolding, polypeptide translocation, and targeted degradation of abnormal and short-lived regulatory proteins.

ClpXP, a well-studied ATP-dependent protease complex from Escherichia coli, is an assembly of homohexameric ClpX coaxially stacked onto a barrel-shaped protease ClpP. ClpX utilizes the energy from ATP hydrolysis to bind the appropriately tagged polypeptide substrates, denature and translocate them into the degradation cavity of ClpP. There have been long-standing questions in the field, specifically whether ClpX can generate force to unfold proteins, how do the six ClpX subunits communicate, and coordinate their ATPases cycles to generate force.

Optical tweezers are used as a powerful single-molecule technique to characterize the mechanochemical properties of biomolecules in the nanometer and piconewton range. After a decade of research, we have developed a novel optical tweezers assay to monitor ClpX as it binds, unfolds and translocates various green fluorescent protein (GFP) fusion substrates in real time under a range of ATP concentrations.

We characterized the general properties of the motor, which are described in chapter 2. From the analysis of these single-molecule trajectories, we have determined several unique properties of the motor previously undetectable in bulk. First, ClpX can generate up to 20 pN of force to translocate and unfold proteins, eventually being stalled in movement as the opposing force approaches 20 pN.

We subsequently explored the communication within the hexameric ring of wild-type ClpX at various ATP, ADP, and Pi concentrations. Our results, described in chapter 3, provided the first direct evidence of a force-generation mechanism and that the phosphate release is coupled to the force-generating step of ClpX. We demonstrated that two to four subunits of the ClpX hexamer actively participates in bursts of translocation and that between the translocation bursts, the motor has a mean constant dwell duration independent of ATP concentration. Our analysis revealed defined a new archetype of motor coordination, which is critical as a fail-safe mechanism to prevent the motor disengagement from its substrate.

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