Student Theses and Dissertations
Quantifying the Release of Protein Substrates from AAA+ ATPase ClpX by Single Molecule Total Internal Reflection Fluorescence Microscopy
Date of Award
Doctor of Philosophy (PhD)
The safe disposal of proteins is an essential process for maintaining proteostasis in cells. AAA+ proteases, such as proteasomes, play a major role in selective degradation of proteins. Degradation by AAA+ proteases typically requires the substrate to be physically unfolded before proteolysis. The efficiency of the unfolding process is hence a critical parameter for determining the turnover rate of the substrate. However, the mechanism by which force is utilized to unfold the protein substrate is not fully characterized. One parameter regulating the efficiency of the unfolding process is the rate by which a substrate is prematurely released before unfolded. Importantly, a propensity to be prematurely released by AAA+ proteases has been proposed to be an important mechanism allowing some substrates to escape proteolysis, but factors that influence the substrate release rate have not been well characterized. In this thesis, we investigate the parameters that influence the premature release of the substrate using the AAA+ ATPase ClpXP. We present a new assay based on Total-Internal-Reflection Fluorescence (TIRF) microscopy that measures the lifetime of the ClpX-substrate complex. We demonstrate that the technique has the potential to identify factors that affect the mean lifetime of the ClpX-substrate complex. Using this method, we show that the substrate release rate can be reduced by simply increasing the length of the unstructured region where ClpX grips the substrate. Next, we demonstrate that reducing the frequency of the ClpX mechanical cycle prolongs the lifetime of ClpX-substrate interaction. We also offer evidence that the association of the protease particle ClpP to the AAA+ particle stabilizes the interaction between ClpX and the protein substrate, even when the substrate does not directly interact with ClpP. Finally, we show that the glycine-alanine repeats, which are associated with proteins that cannot be fully degraded by the proteasomes, do not increase the release rate of the substrate from ClpX. The results indicate that the force ClpX exerts on the substrate can lead to the release of the substrate when it cannot be unfolded. The results support the hypothesis that the transient loss of grip between ClpX and its substrate could be a frequent side-effect of the substrate unfolding process, but ClpX is often able to recover its grip before the substrate dissociates.
Wang, Xiao, "Quantifying the Release of Protein Substrates from AAA+ ATPase ClpX by Single Molecule Total Internal Reflection Fluorescence Microscopy" (2021). Student Theses and Dissertations. 676.
A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy